RAS ASSOCIATED GAP PROTEINS

Abstract: Methods for blocking Ras-induced conditions such as proliferative abnormalities in eukaryote, e.g., mammalian cells. Proteins and mimetics, and their uses, which can block abnormal intracellular signaling often leading to uncontrolled proliferation, e.g., cancers.

Description: RAS ASSOCIATED GAP PROTEINS BACKGROUND OF THE INVENTION Many proliferative cell abnormalities, e.g., cancers, are caused by alterations in the cellular genome. Mutations can affect the expression or function of genes controlling cell growth and differentiation. See, e.g., Bos (1989) πancer Research 49:4682-4689. Examples of such oncogenic mutations include members of the Ras family. See, e.g., Mangues et al. (1992) Seminars in Cancer Research 3:229-239. These genes were initially studied as the viral oncogenes of several transforming retroviruses, and their relationship to cellular counterparts was soon recognized. Genes in the Ras family have been shown to be involved in the transduction of extracellular signals and the control of cellular growth. The Ras family includes three functional genes designated H-ras, K-ras, and N-ras, which encode highly similar proteins. See Barbacid (1987) Ann. Rev. Biochem. 56:779-827. Ras genes from different human tumors were characterized and found to have undergone point mutations leading to constitutive activation, especially codons 12, 13, and 61. These mutant versions are especially potent inducers of tumorigenic or oncogenic transformation. Mutations in the Ras genes may be responsible for as many as 90% of pancreatic adenocarcinomas. The Ras proteins are guanosine triphosphate (GTP)- binding proteins, and serve as a molecular switch in signal transduction controlling the proliferation and differentiation of cells. The linkage of Ras with the nucleoside is non-covalent and designated Ras»GXP to distinguish from a "-" which would indicate a covalent bond. Two different conformational forms of the protein exist depending upon the type of guanine nucleoside attached to the protein. The Ras«GDP form is an inactive form which does not stimulate the downstream effector, e.g., target protein, to result in functional signal transduction. However, the Ras»GTP conformation is active, e.g., stimulates the effector to transmit an activation signal.- Interconversion between the two conformations is enzymatically effected. Conversion from the protein«GDP conformation to the protein«GTP conformation causes activation, and is described as an activation step. Somatic mutations which constitutively activate Ras, e.g., oncogenic Ras, may contribute to tumorigenesis in up to 30% of human tumors. See, e.g., Bos (1989) Cancer Res. 49:4682- 4689; and Rodenhuis (1992) Seminars in Cancer Biol. 3:241-247. Most anti-cancer drugs currently available are not directed toward specific oncogenes, but rather inhibit even normal cellular processes. These drugs are non-specific and cause severe side-effects, e.g., killing any and all proliferating cell types. Many of these proliferating cells are important for sustaining the organism, e.g., the hematopoietic and immune systems and the intestinal lining. Treatment for proliferative cell conditions, e.g., chemo- or radio-therapy have debilitating side effects due to the nonspecificity of the drugs. A need exists for means to more directly target therapeutic reagents to the proper abnormal cells. The next generations of anti-cancer drugs will be compounds which specifically target particular oncogenes, e.g., Ras. Thus, the development of anti-cancer drugs specifically targeting Ras oncogenes is an important goal to conquer human malignancies. The present invention provides these and many other advantages. SUMMARY OF THE INVENTION The present invention provides methods for blocking Ras-induced effects on eukaryotic cells. Different Ras mutations have been demonstrated to cause oncogenic transformation in eukaryotic cells by providing constitutive activation signaling to the cells. Various fragments of GTPase Activating (GAP) proteins have been identified which specifically interact with defined Ras mutants to block signal transduction. These fragments likely function through a mechanism of interacting with the Ras»GTP activated conformation to block the natural interaction of the effector protein. These fragments thus block the constitutive signal transduction which results in Ras induced constitutive effects. The present invention provides methods of blocking a Ras-induced effect on a cell, comprising a step of introducing a GTPase Activating (GAP) protein to the cell. Ordinarily, the Ras will be an oncogenic Ras or one which substantially lacks GTPase activity. The Ras-induced effect will typically be induction of cell proliferation or transformation. The cell will often be eukaryotic cell, e.g., a mammalian cell, including a human cell. On some embodiments, the step of introducing is by expression of a nucleic acid encoding the GAP protein. In preferred embodiments, the GAP protein will bind to the Ras protein with a Kd of less than 200 nM. In other embodiments, the GAP protein is selected from: (a) a fragment of a mammalian GAP protein; (b) a fragment of a mammalian NF1-GRD protein; and (c) a homologue or mimetic of (a) or (b) . In particular embodiments, the GAP protein is selected from: (a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and (b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position 1063 through 1651 of NF1 or the corresponding region of other GAP proteins. Many of these substitutions will be a conservative substitution. In other embodiments, the GAP protein will interact with Ras and block interaction of an effector molecule which binds to Ras at a position corresponding to a position from 32 to 40 or from 59 to 65. In various preferred embodiments, the GAP protein does not block signal transduction of non-oncogenic Ras. Greater specificity of action can be achieved by identifying the responsible oncogenic Ras and selecting a GAP protein which specifically blocks the identified oncogenic Ras. The invention also provides methods of treating an oncogenic Ras transformed cell comprising the step of introducing to said cell a GAP protein capable of suppressing the transformation of said cell. Often, the oncogenic Ras transformed cell will be a mammalian cell, including a human cell. In some embodiments, the GAP protein does not block signal transduction of non-oncogenic Ras. The method can be improved by adding steps of identifying the responsible oncogenic Ras and selecting a GAP protein which blocks transformation by the identified Ras. Preferably, the GAP protein does not block signal transduction of non-oncogenic Ras, e.g., exhibiting specificity. In addition, the invention provides methods of identifying appropriate GAP proteins useful for treating a mutated Ras-induced condition of a eukaryote cell comprising: (a) identifying the mutated Ras which induces the condition; and (b) screening various GAP variants for specific variants which are capable of blocking the condition. In some embodiments, the eukaryote cell is a mammalian cell, including a human cell. In a preferred embodiment, additional screening is performed to determine which GAP variants have minimal effect on non-mutated Ras effects. The invention further provides GAP proteins capable of blocking transformation of a cell, where said transformation is due to an oncogenic Ras. In some cases, the GAP protein is selected from: (a) a fragment of a mammalian GAP protein; (b) a fragment of a mammalian NFl- GRD protein; and (c) a homologue or mimetic of (a) or (b) . In others, the GAP protein is selected from: (a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and (b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position from 1063 through 1651 of NFl or the corresponding region of other GAP proteins. Often the substitution will be a conservative substitution. In other embodiments, the protein interacts with Ras and blocks interaction of an effector molecule which binds to Ras at a position from 32 to 40 or from 59 to 65. Often the cell iε a eukaryotic cell, e.g., a mammalian cell, including a human cell. In preferred embodiments, the oncogenic Ras substantially lacks GTPase activity. In other embodiments, the protein binds to oncogenic Ras with a Kd of less than 200 nM. Mechanistically, the protein may interfere with interaction of Ras»GTP with an effector compound. In another embodiment, the invention provides an isolated nucleic acid encoding a protein normally expressed as a protein as described. BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows stimulation of GTPase activity of c-Ha- Ras Gl 12 and c-Ha-Ras Va112 proteins by yeast cell extracts containing wild-type and mutant NFl-GRDs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OUTLINE I. Ras family A. structure and function B. cycling between Ras•GDP and Ras»GTP II. GAP proteins; family, mammalian, NFl III. Interaction of Ras and GAP proteins IV. Downstream signal transduction V. Methods A. administering B. matching to corresponding Ras C. making compositions, analogues, mimetics I. Ras family A. structure and function Ras gene family members are ubiquitous among eukaryotic cells. See, e.g., Barbacid (1987) Ann. Rev. Biochem. 56:779-827. The genes were initially identified and studied as the viral oncogenes of several acute transforming retroviruses. The relationship to human cancer was quickly established upon recognition that the retroviral oncogenes were derived from a group of mammalian cellular proto-oncogenes, e.g., endogenous genes which become oncogenic upon mutation. Point mutations in the normal endogenous mammalian Ras gene often led to an oncogenic transformed phenotype. Further studies on the locations of the point mutations showed a high frequency at particular hot spots, e.g., codons 12, 13, or 61. Recent technology, e.g., selective hybridization with specific probes, and PCR techniques have simplified analysis of specific alterations responsible for Ras-induced effects. See Bos (1988) Mutat. Res. 195:255- 271. Extension of interest to the counterparts in non- mammalian systems has shown that these genes play a critical role in transduction of many extracellular signals in cells. Functional and structural data has shown that Ras proteins are GTP-binding proteins involved in transduction of signals in response to extracellular stimuli. The family of Ras proteins can be defined by a combination of functional and structural criteria. See, e.g., Bollag et al. (1991) Ann. Rev. Cell Biol. 7:601-632. Ras-induced effects are the functional consequences of Ras activation. In mammalian cells, typically the Ras-induced effects will be cell transformation, but may also include differentiation or proliferation effects which fail to satisfy the full criteria for transformation. The yeast Saccharomvces cerevisiae possesses two members of the Ras family (Rasl and Ras2) which play an important role in cell growth through the regulation of adenylate cyclase. See, e.g., Broach et al. (1990) Adv. Cancer Res. 54:79-139. The Ras-induced effects in yeast show a heat-shock sensitive phenotype. Members of the Ras family have also been studied in Xenopus laevis; Drosophila melanogaster, Caenorhabditis elegans; and Dictyostelium discoideum. See Bollag et al. (1991) Ann. Rev. Cell Biol. 7:601-632; and Kaziro et al. (1991) Ann. Rev. Biochem. 60:349-400. Although the Ras-induced effects may be different in different cells, the relationship in structure often allows cross species interactions of corresponding proteins in Ras related pathways. Exploitation of these structural similarities provide useful means to test interaction of proteins which normally are never found together with advantages directed towards ease of testing effects on various cell sources. B. cycling between Ras*GDP and Ras»GTP The Ras proteins have been shown to be GTP-binding proteins. They can be either in GDP-bound conformation or a GTP-bound conformation. The GTP-bound conformation is the active and interacts with an as yet unidentified effector molecule. Current models propose that Ras proteins become activated upon stimulation, transduce the signal to an as yet unidentified effector molecule, and subsequently become inactivated. Mutated, e.g., oncogenic, Ras proteins have lost their ability to become inactivated and thus constitutively send a stimulation signal. Ras is active in its GTP-bound form. -The active Ras»GTP complex, which is a non-covalent association, is converted to an inactive Ras-guanosine diphosphate (Ras»GDP) form by an intrinsic GTPase activity found on normal Ras, and which is stimulated by a GTPase Activating (GAP) protein. However, oncogenic Ras lacks the intrinsic GTPase activity and GAP proteins have little, if any, effect on inactivating oncogenic Ras. This substantial lack of GTPase activity in oncogenic Ras will typically be at least 20% less than the normal, more typically at least 35% less, usually at least 50%, more usually at least 60% less, preferably at least 70% less, and more preferably at least 80% or more less than normal Ras. II. GAP proteins; family, mammalian, NFl GTPase activities are required to inactivate the Ras»GTP form of the protein in the cycling reaction. A family of proteins stimulating endogenous GTPase activities of Ras proteins have been described which share structural and functional similarities. See Bollag et al. (1991) Ann. Rev. Cell Biol. 7:601-632. Particularly relevant members of the GAP family include yeast and mammalian proteins, including the human neurofibromatosis type 1 (NFl) protein. As used herein, GAP protein refers to a protein which shares structural or functional properties with this family of proteins. Usually, the protein will be a fragment shorter than the natural mammalian proteins so far described, normally less than about 600 amino acids, more normally less than about 550 amino acids, ordinarily less than about 500 amino acids, more ordinarily less than about 460 amino acids, usually less than about 420 amino acids, more usually less than about 380 amino acids, typically less than about 350 amino acids, more typically less than about 325 amino acids, preferably less than about 310 amino acids, more preferably less than about 300 amino acids, and in other embodiments, even fewer amino acids, down to 200 or fewer amino acids. NFl was first identified as the gene responsible for the pathogenesis of the human genetic disorder, neurofibromatosis type 1. cDNA cloning revealed that the NFl gene encodes a protein of 2818 amino acids. This putative protein product has a domain showing a significant sequence homology with members of the Ras GTPase-activating protein (GAP) family. See, e.g., Gutmann et al. (1992) Ann. Neurol. 31:555-561; Xu et al. (1990) £_U 63:835-841; Martin et al. (1990) Cell 63:843-849; and Ballester et al. (1990) Cell 63:851-859. This domain, a fragment of the natural NFl, is often referred herein as NFl GAP Related Domain (NFl-GRD) , and some fragments thereof should have similar activities. Two yeast Saccharomvces cerevisiae proteins, Iral and Ira2, show particularly high sequence homology to the NFl. Subsequent studies have demonstrated that members of the GAP family, including the GAP-related domain of the NFl gene product (NFl-GRD; sometimes referred to as NFl fragment) , can stimulate guanosine triphosphatase (GTPase) activity of Ras proteins, i.e., converting Ras«GTP to Ras»GDP, and thereby negatively regulate the activity of Ras. Two proteins which regulate the activity of Ras proteins are the GTPase activating protein (GAP) and the protein encoded by NFl, the gene responsible for neurofibomatosis. type I disease. See Gutmann et al. (1992) Ann. Neurol. 31:555-561. III. Interaction of Ras and GAP proteins The GAP proteins have been identified as one of the means by which activated Ras proteins are converted into the inactive form. Thus, the physical interaction of the GAP and Ras proteins are important in the understanding of the functional relationship between the entities. The GAP protein effect on endogenous GTPase activity of RAS has been localized to a fragment of the natural GAP protein, e.g., wild-type sequences. In particular, the catalytic domain has been localized to the carboxy terminal segment of the mammalian GAP proteins. The active portion has been localized to a fragments of less than about 600 amino acids, corresponding to the NFl amino acids 1063- 1651. As such, the functional activities of the GAP proteins would be expected to be localized in this region of the sequence. The sites of GAP interaction with Ras have been proposed to be positions 32-40 and 59-63 of mammalian Ras. The yeast S. cerevisiae possesses two NFl homologues, Iral and Ira2. The human NFl is structurally closer to yeast Ira than human GAP and thus would be expected to interact well with the yeast Ras counterpart proteins. This structural similarity is reflected in a functional relationship, as NFl-GRD expressed in yeast cells can complement ira-deficient yeast. In ira ~ cells, the conversion of Ras*GTP to Ras*GDP is defective, and the cells show a phenotype which is very similar to that of activated Ras mutants, i.e., heat shock-sensitivity. The GAP-Related Domain of the NF-1 gene product (NFl-GRD) is a fragment from the NF-1 which can suppress the heat- sensitive phenotype of ira ~ , but not of RAS2 Va H9 or RAS 2Leu68. This is consistent with the fact that NFl-GRD stimulates GTPase activity of normal but not mutant Ras proteins. Thus, the natural GAP will have blocking effects of Ras functions of normal cells. IV. Downstream signal transduction The biochemical mechanism of signal transduction, or effect, of Ras activation is poorly understood. The structural means by which signal transduction occurs has not been clarified, but it is believed that an effector compound, likely a protein, interacts with Ras»GTP. Genetic analysis of the amino acid positions which affect effector binding have been postulated to include positions 32, 35, 36, 38 and 40. Thus, the effector may well bind near to the same sites of Ras as does the GAP proteins. This has led to the model that variants of GAP segments may interact with Ras in a fashion which can block effector interaction. This will function to block signal transduction, in a fashion which will inactivate an oncogenically transformed Ras. Moreover, since the different oncogenic Ras forms result from mutations at sites near the GAP and effector interaction sites, variant GAP segments may show great specificity in blocking Ras- induced effects. In particular, the binding affinity of the GAP analogues which block Ras-induced effects are higher than normal GAP binding. In particular embodiments, the GAP protein, which is intended here to also encompass the concept of protein analogues and mimetics, will preferably be a relatively small polypeptide or analogue, including modified proteins and mimetics. Mimetics include compounds possessing similar molecular shapes sufficient to confer the desired biological property. Various amino acid substitutions may be designed, tested, or screened for activity in blocking Ras-induced functions. These may be effective in blocking effects of many different Ras mutants, or specific Ras variants. The methodology described herein may be useful to define GAP proteins which exhibit high specificity for only interacting with oncogenic, e.g., mutant Ras, and having virtually no effect on natural Ras function. Thus, the GAP proteins provided herein will be highly specific in affecting only oncogenic functions and will be innocuous in cells possessing normal Ras. Although the positions of GAP believed to be most important in the interaction with Ras are in the regions of 701-1047 of GAP, the NFl regions considered most likely to be useful herein will be within the region of 1063-1651 or the corresponding region of other GAP proteins, including 1175-1534, and more specifically in the regions of 1400- 1500. Mutations within this region are likely to interact with the Ras in the desired way, particularly in the region of 1421-1461 of NFl or the corresponding region of other GAP proteins. Functionally, the useful GAP proteins have high binding affinity for Ras or Ras-like proteins or GAP binding segments thereof. Typically, the GAP protein will exhibit a Kd for Ras, or its oncogenic variant, of less than about 300 nM, more typically less than about 250 nM, usually less than about 200 nM, more usually less than about 150 nM, preferably less than about 100 nM, and more preferably even higher binding affinity. Typically a higher binding affinity will allow effective competitive effect on the effector binding at low concentrations of GAP protein. IV. Methods A. administering As deεcribed, blocking Ras-induced effects will occur upon proper εelection of the GAP protein, e.g., fragments, analogues, and mimetics, and administering such composition to the cell. The GAP protein will be produced, e.g., by recombinant means, as are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, CSH, N.Y., and Ausubel (1987 and periodic supplements) Current Protocols in Molecular Bioloσv Greene/Wiley, New York; which are each incorporated herein by reference. The GAP protein can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active ingredientε, e.g., in conventional pharmaceutically acceptable carrierε or diluentε, along with phyεiologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. Drug screening using Ras or fragments thereof can be performed to identify compounds having binding affinity. Subsequent biological assays can then be utilized to determine if the compound has intrinsic activity and is therefore a blocker or antagonist in that it blocks the effects of oncogenic Ras. Additional compounds may be screened or designed using the reagents described, or by molecular modeling and structural studies including, e.g., X-ray crystallography, multidimensional NMR, and other techniques. See, e.g., Blundell et al. (1976) Protein Crystallography Academic Press, New York. The quantities of reagents necesεary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosageε used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doseε for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Penn. ; each of which iε hereby incorporated herein by reference. Methodε for adminiεtration are discusεed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and otherε. Pharmaceutically acceptable carrierε will include water, saline, buffers, and other compoundε deεcribed, e.g., in the Merck Index. Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 100 mM concentrations, typically less than about 10 mM concentrations, usually less than about 100 μM, preferably less than about 10 μM, and most preferably less than about 1 μM, with an appropriate carrier. Slow release formulations, or slow releaεe apparatus will often be utilized for continuous administration. The GAP protein may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteinε such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneouε, intramuscular, intravenous, and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al. (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed. , Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Perm.; each of which iε hereby incorporated herein by reference. The therapy of this invention may be combined with or used in asεociation with other chemotherapeutic or chemopreventive agentε. Iεolation and characterization of these nucleic acids allow use thereof to make variants and mutants. It will also allow production of vector conεtructs useful, e.g., for gene therapy. See, e.g., Goodnow (1992) "Tranεgenic Animals" in Roitt (ed.) Encyclopedia of Immunology Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn et al. (1991) Science 254:707-710; Capecchi (1989) Science 244:1288: Robertson (1987) (ed.) Teratocarcinomas and Embryonic Stem Cell;.: A Practical Approach IRL Press, Oxford; and Rosenberg (1992) >L_. Clinical Oncology 10:180-199; which are each incorporated herein by reference. B. matching to corresponding Ras In particular, the present invention allows for simple matching of a therapeutic agent to various oncogenic Ras variants. This can provide highly εelective treatment of defined oncogenic conditionε with a GAP having highly εelected εafety and efficacy combinationε, virtually tailored to the relatively small number of oncogenic Ras mutations which cause defined proliferative conditions. For example, common variants of oncogenic Ras can be used to screen for GAP fragments which are effective in blocking the oncogenic effects. See, e.g. Kumar et al. (1990) Cancer Res. 52:6877-6884. Either the variants or equivalents thereof can be transformed into a cell, e.g., a yeast cell, and GAP mutants teεted for their specific effect on the Ras variants. Once appropriate GAP proteins are identified for each of the common oncogenic Ras mutants, therapeutic reagents can be selected based upon the diagnosed mutant oncogenic Ras reεponεible for a given abnormality. Diagnosis of the responsible Ras mutation can be performed as described above. C. making compositions, analogues, mimetics Isolated GAP encoding DNAε can be readily modified by nucleotide substitutionε, nucleotide deletionε, nucleotide inεertionε, and inverεionε of nucleotide stretches. These modifications result in novel DNA sequences which encode these modified GAP proteins, their derivativeε, or proteinε having the deεired anti- oncogenic activity. Theεe modified sequences can be used to produce mutant GAP proteins or to enhance the expression of GAP. Enhanced expression may involve gene amplification, increased transcription, increased.... translation, and other mechanisms. Such mutant Ras or GAP derivatives include predetermined or site-specific mutations of the respective protein or its fragments. A mutant GAP is a polypeptide otherwise falling within the homology defined by structure and function, but having an amino acid sequence which differε from the corresponding segment of GAP as found in nature, whether by way of an amino acid deletion, subεtitution, or inεertion. Similar proteinε and nucleic acidε should be available from other warm blooded animals, e.g., mammals and birds. These descriptionε are generally meant to encompaεε εpecies and allelic variants of the GAP proteins, not limited to the specific embodiments discuεsed. Although εite εpecific mutation εiteε are predetermined, mutants need not be site specific. GAP protein or Ras protein mutagenesis can be conducted by- making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include but are not limited to amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expresεed GAP mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesiε. See also Sambrook et al. (1989) and Ausubel et al. (1987 and Supplements) . The mutations in the DNA normally should not place coding sequenceε out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such aε loopε or hairpinε. The preεent invention alεo provideε recombinant proteinε, e.g., heterologouε fuεion proteinε uεing εegmentε from these proteins. A heterologous fusion protein is a fusion of proteinε or segments which are naturally not normally fused in the εame manner. Thuε, the fuεion product of an immunoglobulin with a GAP polypeptide iε a continuous protein molecule having sequenceε fused in a typical peptide linkage, e.g., typically made as a single translation product and exhibiting properties derived from each source peptide. A similar concept applies to heterologouε nucleic acid εequenceε. In addition, new conεtructε may be made from combining εimilar functional domainε from other proteins. For example, Ras-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham et al. (1989) Science 243:1330-1336; and O'Dowd et al. (1988) J. Biol. Chem. 263:15985-15992, each of which is incorporated herein by reference. Thus, new chimeric polypeptideε exhibiting new combinationε of specificities will result from the functional linkage of Ras-binding specificities. For example, the Ras-binding segmentε from other related proteinε may be added or combined with other binding segments from other proteins. The resulting protein will often have hybrid function and properties. The phosphoramidite method deεcribed by Beaucage and Carutherε (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragmentε. A double εtranded fragment will often be obtained either by εynthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand uεing DNA polymeraεe with an appropriate primer sequence. The present invention provides means to produce fusion proteins. Variouε GAP variantε may have εlightly different functionε or biological activitieε, even though they share significant structural similaritieε. Dissection of structural elements which effect the various physiological functions or biological activities provided by the GAP proteins is possible using standard techniques of modern molecular biology, particularly in comparing variants within the related family of GAP proteins. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham et al. (1989) Science 243:1339-1336; and approaches used in O'Dowd et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter et al. (1990) EMBO J. 9:4381-4390; each of which is incorporated herein by reference. In particular, Ras binding segments can be subεtituted between proteinε to determine what εtructural featureε are important in both Raε binding affinity and εpecificity for the natural or oncogenic Ras. An array of different Ras variants, e.g., allelic, will be used to screen for GAP proteins exhibiting desired properties of interaction with them, e.g., high binding affinity, blocking of effector function by conformational or competitive inhibition, or even forms which can induce GTPase action of the oncogenic Ras. The specific segments of interaction of GAP with Ras may be identified by mutagenesiε or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will alεo be applicable. Identification of the εimilarities and differences between Ras oncogenic variants will lead to new diagnostic and therapeutic reagents or treatmentε. Structural εtudieε of the Raε variantε will lead to design of new GAP proteins, particularly analogues exhibiting desired effect blocking properties. This can be combined with εcreening methods to isolate new GAP proteins exhibiting desired spectra of activitieε. Both the naturally occurring and the recombinant formε of Raε are particularly uεeful in kitε and assay methods which are capable of εcreening compoundε for binding activity to them. Several methodε of automating aεεays have been developed in recent years so as to permit screening of tens of thousandε of compoundε per year. See, e.g., Fodor et al. (1991) Science 251:767-773, which is incorporated herein by reference and which describes means for testing of binding affinity by a plurality of defined polymers syntheεized on a εolid εubεtrate. Phage or other librarieε of variouε random polypeptide sequences would also be useful. The development of suitable asεayε can be greatly facilitated by the availability of large amountε of purified, soluble Ras, either natural or oncogenic, by methods aε provided herein. Expreεεion in other cell typeε will often reεult in glycoεylation differences in a particular GAP protein. Various mutants may exhibit distinct biological activities based upon structural differences other than amino acid sequence. Differential modifications may be responεible for differential function, and elucidation of the effectε are now made poεεible. A nucleic acid which encodes a Ras and GAP are readily available, or can be obtained by chemical εyntheεiε, εcreening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g., Marchuk et al. (1991) Genomics 11:931-940; and nucleic acid and protein data bases, e.g., Protein Identification Resource (PIR) , Georgetown University, Washington, D.C., SwissProt and otherε, see intelliGenetics, Menlo Park, CA, or the Univ. Wiεconεin Biotechnology Center, Madison, Wisconεin. Thiε DNA can be expreεεed in a wide variety of hoεt cells for the syntheεiε of a Raε, GAP. or fragmentε thereof which can in turn, for example, be uεed to generate polyclonal or monoclonal antibodies; for construction and expression of modified Raε or GAP moleculeε; and for structure/function εtudieε. Each GAP can be expreεεed in host cells that are transformed or transfected with appropriate expreεεion vectorε. These molecules can be subεtantially free of protein or cellular contaminantε, other than thoεe derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent. The GAP, or portions thereof, may be expresεed aε fuεionε with other proteinε. Expreεεion vectorε are typically εelf-replicating DNA or RNA conεtructε containing the deεired Raε or GAP gene or itε fragmentε, uεually operably linked to εuitable genetic control elementε that are recognized in a εuitable hoεt cell. Theεe control elements are capable of effecting expresεion within a suitable host. The specific type of control elements necessary to effect expresεion will depend upon the eventual host cell used. Generally, the genetic control elements can include a prokaryotic promoter syεtem or a eukaryotic promoter expreεεion control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodeε a suitable ribosome binding site, and sequenceε that terminate transcription and translation. Expresεion vectorε also usually contain an origin of replication that allows the vector to replicate independently of the host cell. The vectors of this invention contain DNA which encodes a uεeful GAP-like peptide, or a fragment thereof encoding, e.g., an active polypeptide. The DNA can be under the control of a viral promoter and can encode a εelection marker. This invention further contemplates uεe of such expresεion vectorε which are capable of expreεεing eukaryotic cDNA coding for a GAP in a prokaryotic or eukaryotic hoεt, where the vector iε compatible with the hoεt and where the eukaryotic cDNA coding for the GAP iε inεerted into the vector such that growth of the host containing the vector expresεeε the cDNA in queεtion. Uεually, expreεsion vectors are designed for stable replication in their hoεt cellε or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is poεεible to effect tranεient expresεion of the GAP in variouε hoεtε uεing vectors that do not contain a replication origin that is recognized by the host cell. It is alεo poεεible to uεe vectorε that cauεe integration of GAP into the hoεt DNA by recombination. Vectors, as used herein, comprise plaεmids, viruseε, bacteriophage, integratable DNA fragmentε, and other vehicleε which enable the integration of DNA fragmentε into the genome of the hoεt. Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual. Elsevier, N.Y. , and Rodriguez et al. (edε) Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Buttersworth, Boston, 1988, which are incorporated herein by reference. For purposeε of thiε invention, DNA εequenceε are operably linked when they are functionally related to each other. For example, DNA for a preεequence or εecretory leader is operably linked to a polypeptide if it is expresεed aε a preprotein or participateε in directing the polypeptide to the cell membrane or in εecretion of the polypeptide. A promoter is operably linked to a coding sequence if it controlε the tranεcription of the polypeptide; a riboεome binding site is operably linked to a coding εequence if it iε poεitioned to permit tranεlation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but εtill bind to operator sequences that in turn control expression. Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes include yeaεts, e.g., S. cerevisiae and Pichia. and specieε of the genus pictyostelium. Higher eukaryotes include establiεhed tisεue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents. Prokaryotic host-vector systemε include a wide variety of vectors for many different specieε. As used herein, E. coli and itε vectors will be used generically to include equivalent vectorε used in other prokaryotes. A representative vector for amplifying DNA is pBR322 or many of its derivatives. Vectors that can be used to express the GAP protein include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN-εeries); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540) . See Broεius et al. (1988) "Expresεion Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds. Rodriguez and Denhardt) , Buttersworth, Boston, Chapter 10, pp. 205-236, which is incorporated herein by reference. Lower eukaryotes, e.g., yeasts and Dictvostelium. may be transformed with GAP sequence containing vectorε. For purposes of this invention, the most common lower eukaryotic host iε the baker'ε yeaεt, Saccharomvceε cereviεiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available. Yeast vectors typically consist of a replication origin (unlesε of the integrating type) , a selection gene, a promoter, DNA encoding the Raε or GAP protein or itε fragmentε, and εequences for translation termination, polyadenylation, and transcription termination. Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or εuch inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-εerieε) , εelf-replicating high copy number (such aε the YEp-series); integrating typeε (such as the Yip-series) , or mini-chromosomeε (εuch aε the YCp-εerieε) . Higher eukaryotic cells grown in tissue culture are often the preferred host cells for expresεion of the GAP protein. In principle, any higher eukaryotic tissue culture cell line is workable, e.g., insect baculovirus expreεsion εystems, whether from an invertebrate or vertebrate εource. However, mammalian cells are often preferred. Transformation or transfection and propagation of such cells haε become a routine procedure. Exampleε of uεeful cell lineε include HeLa cellε, Chinese hamεter ovary (CHO) cell lineε, baby rat kidney (BRK) cell lines, insect cell lineε, bird cell lineε, and monkey (COS) cell lines. Expresεion vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used) , a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expresεion vectorε may be plasmids, viruses, or retroviruseε carrying promoterε derived, e.g., from εuch sources as from adenovirus, SV40, parvoviruεes, vaccinia virus, or cytomegaloviruε. Representative exampleε of εuitable expreεεion vectorε include pCDNAl (Invitrogen, San Diego, CA) ; pCD, see Okayama et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, εee Thomaε et al. (1987) ςell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610. It may be desired to express a GAP polypeptide in a system which provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system. For example, the GAP gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells. The broad scope of this invention is best underεtood with reference to the following exampleε, which are not intended to limit the invention in any manner. In theεe studieε, a yeaεt Raε system was used to isolate NFl-GRD mutants which can suppresε specifically the activity of oncogenic Raε. Yeast cells carrying activated mutations in Ras (such as RAS2 Va119 and Δ__2. Leυ68 ) are defective in reεponding to environmental conditionε, and show a variety of phenotypes including a heat shock- sensitive phenotype. First, a pool of randomly mutagenized NFl-GRD genes were screened to obtain suppreεεorε of a εpecific yeaεt oncogenic-type Ras, RAS2 Va l 1 9. Next, these mutant NFl-GRDε were εhown to be capable of inhibiting v-Ras-induced transformation in mammalian cellε. These results demonstrated that this unique yeaεt method provideε a powerful εcreening εystem to obtain anti-Ras NFl-GRD mutantε. The mutantε of NFl-GRD moεt likely bind tightly with the oncogenic, e.g., mutated, Raε proteins to sequester the latter proteins from the signal transduction for normal cell growth. Detailed analysis of the εtructures involved in the interaction between mutant NFl- GRDε and Ras will enable testing of compounds, e.g., analogues and mimetics, which can mimic the action of NFl- GRDs, and inhibit specifically transforming Ras activity. EXAMPLE 1: Preparation of oKPll A plasmid pKPll, which expresεes a domain of NFl (amino acid residues 1063-1651; the numbers of amino acid residues are referred to according to Marchuk et al. (1991) Genomics 11:931-940, and a yeast strain carrying RAS2 Va119 mutation were used to obtain mutant NFl-GAP Related Domains (GRDs) which can εuppreεε the phenotype of activated Raε. In a previouε study, this plasmid was shown to suppreεε ira2 " but not RAS2 Va119 . The plaεmid waε randomly mutagenized by treatment with hydroxylamine in vitro, and a pool of mutagenized DNAs was transformed into RAS2 Va ^- 19 cells. Subsequently, about 2 x 10^ independent colonies were screened for heat shock resistance. Wild-type NFl-GRD was cloned into the yeast expresεion vector pKTlO which contains glyceraldehyde-3-phosphate dehydrogenese promoter, a replication origin derived from 2 μm, and URA3 aε a selection marker to yield pKPll. One hundred micrograms of pKPll DNA was mutagenized by hydroxylamine in vitro as described previously (Roεe et al. (1987) Cell 48:1047-1060), and tranεformed into a _ 2 __ cereviεiae εtrain, TK161-R2V-D which carrieε RAS2 Va119 mutation. See Tanaka et al. (1989) Mol. Cell. Biol. 9:757- 768; and Tanaka et al. (1990) Mol. Cell. Biol. 10:4303- 4313. About 2 x 10^ colonies were grown on selection plates, and the plates were heated at 57 'C for 15 minutes. The resultant plates were incubated at 30 'C for 4 days, and growing colonies were selected. The heat shock- sensitivity of these colonies were checked, and 12 clones were selected at this stage. Plasmid DNAs were recovered from these cellε, re-tranεformed into TK161-R2V-D, and phenotypic reverεion waε examined. Twelve positive colonies were obtained in the initial screening. Subsequently, two clones, NF201 (SEQ ID NO: 1) and NF204 (SEQ ID NO: 2), which had a relatively strong suppression activity for R S2 Va ^ 19 , were selected, and subjected to further analysis. EXAMPLE 2; Effect of Mutant NFl-GRDs on veast cells The effects of NF201 (SEQ ID NO: 1) and NF204 (SEQ ID NO: 2) were tested on different alleles of activated RA 2 V 119 in yeaεt cellε (Table 1) . Wild-type NFl-GRD could weakly revert the phenotype of RAS2 Leu ^8, fc> u t was totally ineffective on RAS2 Va119 and RAS2 Ser41 . Mutant NF201 εuppressed the heat shock-sensitive phenotype of all three alleles of RAS2 examined, including RAS2 Va ^-^ 9 . RA£2 Leu68 , and RA£2 Ser41 (Tanaka et al. (1992) Mol. Cell. Biol. 21:631-637). On the other hand, NF204 preferentially suppressed RAS2 Va l 19 but not the other two alleles . These results indicate that NF201 and NF204 possess distinct properties as suppressors of activated Ras in a Ras- specific manner. Table 1. Suppression of the heat-sensitive phenotypes of various activated alleles of RAS2 by mutant NFl-GRD. A wild-type S. cerevisiae εtrain, RAY-3A-D, harboring a combination of RAS2 plasmids (YCp-__A___L Va119 , -EΔ__2 Leu68 , and -£______ Ser41 Tanaka et al. (1992) Mol. Cell. Biol. 12:631-637) and NFl-GRD plasmidε, waε εubjected to heat shock asεay. The ability of each NFl-GRD plaεmid to εuppreεε the heat-εensitive phenotype was scored: +++, strong suppression; ++, intermediate suppression; +, weak suppresεion; -, no detectable εuppreεεion. The complementation activity in ira2 " cells (KT63-2B-D; Tanaka et al. (1989) Mol. Cell. Biol. 9:757-768; Tanaka et al. (1990) Mol. Cell. Biol. 10:4303-4313), which reflects the activity of these NFl-GRDs on wild-type RAS2 (RAS2 wt ) . waε alεo εcored, and iε εhown in the table. Intereεtingly, these two mutant NFl-GRDs could suppresε ira2 ~ cells, in which normal Ras proteins are activated, to the same extent as wild-type NFl-GRD, suggeεting that NF201 and NF204 retain the ability to εtimulate GTPase activity of normal Ras. The entire region of mutant NFl-GRDs were sequenced to identify mutations in NF201 and NF204, and the εequenceε compared the εequenceε with that of wild-type NFl-GRD. In both NF201 and NF204, εingle nucleotide changes were found in the DNA sequences. In NF201 (SEQ ID NO: 1), the codon TT£ for Phe at residue 1434 was changed to TTA coding for Leu, while in NF204 (SEQ ID NO: 2), the codon Aj__G for Lys at residue 1436 was replaced by AG.A coding for Arg. Although both mutation siteε are located in one of the moεt conserved regions of the GAP-related domain (see Xu et al. (1990) Cell 63:835-842; Martin et al. (1990) Cell 63:843-850; and Ballester et al. (1990) Cell 63:851-859), the amino acid residueε at theεe sites (Phe at position 1434, and Lys at position 1436) are not strictly conserved among the members of the GAP family (Figure 1) . Phe residue at 1434 in NFl is conserved in yeast Ira2 (SEQ ID NO: 4) protein, but it is replaced by other residues in Iral (SEQ ID NO: 3), GAP (SEQ ID NO: 5), and Gapl (SEQ ID NO: 6) . On the other hand, Lys residue at 1346 iε conεerved among NFl, Iral, GAP, and Gapl, but Ira2 containε Arg at the correεponding εite. Recently, two independent εtudieε have demonεtrated that Lyε at poεition 1423 in NFl- GRD, which iε located juεt 11 and 13 amino acids upstream of the mutation sites of NF201 and NF204, respectively, is important for the structure and function of NFl. First, the substitution of Glu for Lys at position 1423 has been identified in some human tumors as well as in a family of neurofibromatosis patients (Li et al. (1992) Cell 69:275- 281) . The GAP activity of this mutant NFl-GRD was 200- to 400-fold lower than that of the wild-type NFl-GRD. It was also reported that the substitution of Met for Lys at the same position resulted in a decrease in stability of the protein (Wiesmuller et al. (1992) J. Biol. Chem. 267:10207- 10219). Thus, the amino acid residues at 1423, 1434 and 1436, and their surrounding sequence, are likely to be important for the structure and/or function of NFl proteins. EXAMPLE 3: Effect of mutant NFl-GRDs in mammalian cells The effect of these mutant NFl-GRDs in mammalian cells was investigated. The cDNA fragments of the wild-type and mutant NFl-GRDs were recloned into a mammalian expression vector, and transfected into cell lineε. The size of the NFl-GRD protein tranεiently expreεεed in Cos7 cells was checked. Western blot analysiε with an anti-NFl-GRD anti-εerum (εee Hattori et al. (1992) Oncogene 7:481-485) identified a protein band of an apparent molecular mass of 67-68 kDa in the cells transfected with NFl-GRD plasmids but not with the control vector. This suggeεtε that the protein of about 67 kDa waε tranεlated εtarting from the internal Met reεidue at poεition 1073 of NFl CDNA. The anti-Raε activitieε of mutant NFl-GRDs were examined for their effects on v-Raε-induced tranεformation. The above plasmids expresεing NFl-GRD were cotransfected with pSV2n_e_o_ into DT cells, a v-Ki- £ ≤s_-transformed NIH3T3 derivative, and the ability to induce morphological reversion of the cells was examined. Aε shown in Table 2, transfection of the plasmidε expreεεing NF201 and NF204 could induce flat reversion at dramatically high frequencies (8-9% of total G418-reεiεtant colonieε) . The frequency was even higher than that obtained by transfection of a K∑e^ -1 plasmid which haε been εhown to posseεs anti-oncogenic activity in DT cells (Kitamura et al. (1990) Proc. Natl Acad. Sci. USA 87:4284-4288). Under the same conditions, the wild-type NFl-GRD could also induce flat reversion of DT cells, although it waε 5 to 6 timeε leεε potent than mutant cloneε. This is particularly interesting since a previouε εtudy haε εhown that overexpreεεion of GAP inhibited normal c-Ha-Raε- but not v- Ha-Ras-induced transformation (εee Zhang et al. (1990) Nature 346:754-756) . No revertant of DT cellε could be obtained from tranεfectantε of the GAP plaεmid (Table 2). Thiε difference may be due to the fact that NFl-GRD possesses a much higher affinity for Ras proteins than GAP. These reεultε clearly demonεtrate that mutant NFl-GRDε poεεeεε transformation- εuppressor activity against oncogenic Ras. Table 2. Induction of morphological reversion of v-Ras- tranεformed cellε by mutant NFl-GRD. DT cellε were cotranεfected with 20 μg of NFl-GRD plaεmids and 2 μg of pSV2n_ej_. as described by Kitamura et al. (1990) proc. Natl Acad. Sci. USA 87:4284-4288, and transfectants were selected in a medium containing 0.5 mg/ml G418. Since pKrev-1 plasmid itεelf contained the neo gene, 2 μg of the plaεmid waε cotranεfected with 20 μg of pEF-BOS (the vector for NFl-GRD) . The pEF-GAP contained rat full-length GAP cDNA in pEF-BOS. Frequency of reverεion in DT cellε iε defined aε the ratio (%) of morphologically flat cell colonies to total G418-resistant colonies. N.D. : not determined. Flat colonies/G418-resistant colonies transfected Exp.1 Exp.2 Exp.3 ratio DNA (%) pEF-BOS 0/1155 (<0.1) 2/1279 (0.1) 3/878 (0.4) 0.1 pEF-NFl 20/1522 (1.3) 26/1151 (2.3) 15/1004 (1.5) 1.7 PEF-NF201 86/1190 (7.2) 61/691 (8.8) 34/356 (9.6) 8.0 PEF-NF204 40/448 (8.9) 46/426 (10.8) 24/350 (6.9) 9.0 pEF-GAP N.D. 0/856 (<0.1) 0/561 (<0.2) <0.1 pKrev-1 N.D. 26/1385 (1.9) 15/736 (2.0) 1.9 EXAMPLE 4: Biochemical properties of the mutant. NFl-GRDs The biological properties of the mutant NFl-GRDs were studied to underεtand the molecular mechanism of anti- oncogenic activity. Extracts were prepared from yeast cells expresεing wild-type and mutant NFl-GRDε, and GTPase- εtimulating activity waε meaεured in vitro by uεing recombinant c-Ha-Raε proteinε as subεtrateε. Recombinant c-Ha-Ras G ly!2 (A) or c-Ha-Ras Va l 12 (B) proteinε were loaded with [γ- 32 P]GTP (30 Ci/mmol) in buffer B (50 mM triε-HCl [pH 7.4], 50 mM KCl, 1 mM MgCl2, 2.5 mM EDTA, and 0.2 mg/ml BSA) at 30 'C for 10 minuteε. The reaction was stopped by the addition of MgCl2 to the final concentration of 7 mM. Yeast cell extracts were prepared from wild-type yeast cells, RAY-3A-D, carrying various NFl-GRD plasmids. Cells grown to the stationary phase were collected, and disrupted with acid-washed glasε beadε (0.5 mm diameter) in buffer A (50 mM triε-HCl [pH 7.4], 100 mM KCl, 5 mM MgCl2, 2 mM DTT, 2 mM PMSF, 1 mM benzamidine, and 10 μg/ml of each of pepεtatin A, aprotinin, and leupeptin. The crude extract waε clarified twice by centrifugation at 2000 x g for 20 minuteε. The reεultant supernatants were then mixed with an aliquot of Raε* [γ- 32 p]GTP mixture, and incubated at 30 "C. At the indicated time point, an aliquot was filtered through a nitrocellulose membrane, and radioactivity retained on the membrane was counted. The final concentrations of yeast extract proteinε and Ras* [γ-3 2 p]GTP were 1 mg/ml and 11.5 nM, respectively. The cell extracts assayed were from the cell carrying the following plasmids: ., wild-type NFl-GRD; o, NF201; Δ, NF204; [solid square], vector alone; or [open square] , buffer A plus 1 mg/ml BSA. Two mutant NFl-GRDε, NF201 and NF204, stimulated the GTPase activity of to the εame extent aε wild-type NFl-GRD (Figure 1) . This is consistent with the observation that NF201 and NF204 can effectively complement ira2 " in yeast (see Table 1) . On the other hand, the same extracts were not able to stimulate the GTPase activity of c-Ha-Ras Va l 12 under these experimental conditions. This suggeεtε that the anti- oncogenic activity of the mutant NFl-GRD iε not due to the stimulation of the slow GTPase of oncogenic Ras proteins. The members of the GAP family negatively regulate the activity of Ras by stimulating intrinsic GTPase activity of normal Ras proteins. Thus, NFl can potentially act as a specific block of effector function by normal Ras. However, oncogenic Ras lacks the intrinsic GTPase activity, and thus, natural GAP sequenceε cannot stimulate the inactivation of the activated oncogenic Ras. Likewise, NFl-GRD suppreεεeε the heat shock-sensitive phenotype of ira ~ cells, but not the same phenotype of activated mutants of Ras, e.g., E____2 Va119 and BA__2. eu68 which correspond to mammalian oncogenic Ras, ras Va ^-^- and £ja__ Leu 61, respectively. Various mammalian oncogenic Ras mutants may be simulated by corresponding mutations in yeast Raε proteinε. Theεe obεervationε lead to a model which iε useful for testing interaction of Ras variants with GAP variants, and which predictε uεeful blocking or reversal of mutant or oncogenic Ras-induced effects. A model of anti-oncogenic activity of mutant NFl-GRD consistent with these observations is that the mutant NFl- GRD has higher affinity for oncogenic Ras*GTP as compared to the wild-type NFl-GRD. As discussed above, the GAP binding region, and the effector binding regions on the Ras protein are in cloεe phyεical proximity. Aε εuch, mutant NFl-GRD binding to oncogenic Raε, e.g., high affinity binding, could form an irreversible NFl*Ras*GTP complex. This could prevent interaction with putative downstream effector molecules, e.g., by conformational changes or competition. The oncogenic Ras would be sequestered from signal transduction pathways. Two observations support this hypotheεis. First, as shown in Table 1, weak but significant phenotypic reversion of RAS2 Leu ^8 by wild-type NFl-GRD was obεerved. A previous study (Bollag et al. (1991) Nature 351:576-579) showed that the mammalian Ras Leu61 protein (correεponding to yeast RAS2 Leu ^ 8 ) has a much higher affinity for NFl-GRD than the wild-type or Vall2-form of Raε. The high affinity binding between RAS2 Leu68 and wild-type NFl-GRD can explain the phenotypic εuppression. Likewiεe, thiε model can also explain the differences in transformation-εuppressor activitieε among GAP, wild-type NFl-GRD, and mutant NFl-GRDε. Table 2 shows that wild-type NFl-GRD, but not GAP, can suppreεε tranεformation by v-Raε; two mutant NFl-GRDε are more potent suppresεorε than wild-type NFl-GRD. Thiε order of potency aε tranεformation εuppressors may reflect the relative affinity for Raε proteins; that is, wild-type NFl- GRD has 20 times higher affinity for Ras than GAP (see Martin et al. Cell 63:843-850); mutant NFl-GRDs may have even greater affinitieε. In relation to thiε, it εhould be noted that Ballester et al. (1990) Cell 63:851-859 previously observed the inhibitory effect of wild-type NF1- GRD but not of GAP on c-Ha-Ras Va H 2 expressed in yeast cells. This iε conεistent with the observation that wild- type NFl-GRD can weakly suppress v-Ras-tranεformation in mammalian cellε. The second observation supporting this model is that NF201 can suppress the activity of not only RAS2 Va119 and EΔ21 heu68 , but also E___£2. Ser41 . It has been shown that Ser41 mutation (corresponding to Ser34 of human Ras), which is located in the so-called "effector region," disrupts the effective binding of Ras2 proteinε to yeast Ira proteins as well as NFl-GRD and GAP (Tanaka et al. (1992) Mol. Cell. Biol. 12:631-637). Thus, the fact that NF201 can inhibit the activity of RAS2 Ser41 εtrongly εuggeεtε that the mutation in NF201 reεtores the interaction between RAS2 Ser41 and NFl-GRD. Comparison of the relative affinities of wild-type and mutant NFl-GRDs for oncogenic Raε proteinε εhould provide a teεt for thiε model. Thiε model predictε that highly specific reagents could be produced having specificity only for blocking oncogenic Ras effects while having virtually no effects on normal Ras. In summary, the data presented herein demonstrated that NFl-GRDs with single amino acid εubstitutions can suppress the biological activity of oncogenic Ras. According to the propoεed model, mutant NFl-GRDε could inhibit specifically oncogenic but not normal Raε. In the case of normal Ras*GTP, bound GTP would be rapidly hydrolyzed to GDP upon interaction with NFl-GRD, and NFl- GRD would be released from Ras*GDP. In this study, a mutant NFl-GRD was expreεsed as a protein of 578 amino acidε, which is still a substantially large protein. The yeast screening syεtem described will allow determination of the minimum fragment of NFl-GRD which retainε anti- oncogenic activity. Thiε approach will allow development of Raε-specific anti-oncogenic compounds. All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Schering Corp. (ii) TITLE OF INVENTION: RAS Associated GAP Proteins (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Schering Corp. (B) STREET: 1 Girald Farms (C) CITY: Madison (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP: 94304-1104 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Macintosh (C) OPERATING SYSTEM: 6.0.8 (D) SOFTWARE: Microsoft Word 5.1a (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/004,824 (B) FILING DATE: 15-JAN-1993 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Lunn, Paul G. (B) REGISTRATION NUMBER: 32,743 (C) REFERENCE/DOCKET NUMBER: DX0352 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (201)822-7255 (B) TELEFAX: (201)822-7039 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2485 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 564..9380 ( i) SEQUENCE DESCRIPTION: SEQ ID NO:1: Asn Trp Glu Asp Asn Ser Val lie Phe Leu Leu Val Gin Ser Met Val 1 5 10 15 Val Asp Leu Lys Asn Leu Leu Phe Asn Pro Ser Lys Pro Phe Ser Arg 20 25 30 Gly Ser Gin Pro Ala Asp Val Asp Leu Met lie Asp Cys Leu Val Ser 35 40 45 Cys Phe Arg lie Ser Pro His Asn Asn Gin His Phe Lys lie Cys Leu 50 55 60 Ala Gin Asn Ser Pro Ser Thr Phe His Tyr Val Leu Val Asn Ser Leu 65 70 75 80 His Arg lie lie Thr Asn Ser Ala Leu Asp Trp Trp Pro Lys lie Asp 85 90 95 Ala Val Tyr Cys His Ser Val Glu Leu Arg Asn Met Phe Gly Glu Thr 100 105 110 Leu His Lys Ala Val Gin Gly Cys Gly Ala His Pro Ala lie Arg Met 115 120 125 Ala Pro Ser Leu Thr Phe Lys Glu Lys Val Thr Ser Leu Lys Phe Lys 130 135 140 Glu Lys Pro Thr Asp Leu Glu Thr Arg Ser Tyr Lys Tyr Leu Leu Leu 145 150 155 160 Ser lie Val Lys Leu lie His Ala Asp Pro Lys Leu Leu Leu Cys Asn 165 170 175 Pro Arg Lys Gin Gly Pro Glu Thr Gin Gly Ser Thr Ala Glu Leu lie 180 185 190 Thr Gly Leu Val Gin Leu Val Pro Gin Ser His Met Pro Glu He Ala 195 200 205 Gin Glu Ala Met Glu Ala Leu Leu Val Leu His Gin Leu Asp Ser He 210 215 220 Asp Leu Trp Asn Pro Asp Ala Pro Val Glu Thr Phe Trp Glu He Ser 225 230 235 240 Ser Gin Met Leu Phe Tyr He Cys Lys Lys Leu Thr Ser His Gin Met 245 250 255 Leu Ser Ser Thr Glu He Leu Lys Trp Leu Arg Glu He Leu He Cys 260 265 270 Arg Asn Lys Phe Leu Leu Lys Asn Lys Gin Ala Asp Arg Ser Ser Cys 275 280 285 His Phe Leu Leu Phe Tyr Gly Val Gly Cys Asp He Pro Ser Ser Gly 290 295 300 Asn Thr Ser Gin Met Ser Met Asp His Glu Glu Leu Leu Arg Thr Pro 305 310 315 320 Gly Ala Ser Leu Arg Lys Gly Lys Gly Asn Ser Ser Met Asp Ser Ala 325 330 335 Ala Gly Cys Ser Gly Thr Pro Pro He Cys Arg Gin Ala Gin Thr Lys 340 345 350 Leu Glu Val Ala Leu Tyr Met Phe Leu Trp Asn Pro Asp Thr Glu Ala 355 360 365 Val Leu Val Ala Met Ser Cys Phe Arg His Leu Cys Glu Glu Ala Asp 370 375 380 He Arg Cys Gly Val Asp Glu Val Ser Val His Asn Leu Leu Pro Asn 385 390 395 400 Tyr Asn Thr Phe Met Glu Phe Ala Ser Val Ser Asn Met Met Ser Thr 405 410 415 Gly Arg Ala Ala Leu Gin Lys Arg Val Met Ala Leu Leu Arg Arg He 420 425 430 Glu His Pro Thr Ala Gly Asn Thr Glu Ala Trp Glu Asp Thr His Ala 435 440 445 Lys Trp Glu Gin Ala Thr Lys Leu He Leu Asn Tyr Pro Lys Ala Lys 450 455 460 Met Glu Asp Gly Gin Ala Ala Glu Ser Leu His Lys Thr He Val Lys 465 470 475 480 Arg Arg Met Ser His Val Ser Gly Gly Gly Ser He Asp Leu Ser Asp 485 490 495 Thr Asp Ser Leu Gin Glu Trp He Asn Met Thr Gly Phe Leu Cys Ala 500 505 510 Leu Gly Gly Val Cys Leu Gin Gin Arg Ser Asn Ser Gly Leu Ala Thr 515 520 525 Tyr Ser Pro Pro Met Gly Pro Val Ser Glu Arg Lys Gly Ser Met He 530 535 540 Ser Val Met Ser Ser Glu Gly Asn Ala Asp Thr Pro Val Ser Lys Phe 545 550 555 560 Met Asp Arg Leu Leu Ser Leu Met Val Cys Asn His Glu Lys Val Gly 565 570 575 Leu Gin He Arg Thr Asn Val Lys Asp Leu Val Gly Leu Glu Leu Ser 580 585 590 Pro Ala Leu Tyr Pro Met Leu Phe Asn Lys Leu Lys Asn Thr He Ser 595 600 605 Lys Phe Phe Asp Ser Gin Gly Gin Val Leu Leu Thr Asp Thr Asn Thr 610 615 620 Gin Phe Val Glu Gin Thr He Ala He Met Lys Asn Leu Leu Asp Asn 625 630 635 640 His Thr Glu Gly Ser Ser Glu His Leu Gly Gin Ala Ser He Glu Thr 645 650 655 Met Met Leu Asn Leu Val Arg Tyr Val Arg Val Leu Gly Asn Met Val 660 665 670 His Ala He Gin He Lys Thr Lys Leu Cys Gin Leu Val Glu Val Met 675 680 685 Met Ala Arg Arg Asp Asp Leu Ser Phe Cys Gin Glu Met Lys Phe Arg 690 695 700 Asn Lys Met Val Glu Tyr Leu Thr Asp Trp Val Met Gly Thr Ser Asn 705 710 715 720 Gin Ala Ala Asp Asp Asp Val Lys Cys Leu Thr Arg Asp Leu Asp Gin 725 730 735 Ala Ser Met Glu Ala Val Val Ser Leu Leu Ala Gly Leu Pro Leu Gin 740 745 750 Pro Glu Glu Gly Asp Gly Val Glu Leu Met Glu Ala Lys Ser Gin Leu 755 760 765 Phe Leu Lys Tyr Phe Thr Leu Phe Met Asn Leu Leu Asn Asp Cys Ser 770 775 780 Glu Val Glu Asp Glu Ser Ala Gin Thr Gly Gly Arg Lys Arg Gly Met 785 790 795 800 Ser Arg Arg Leu Ala Ser Leu Arg His Cys Thr Val Leu Ala Met Ser 805 810 815 Asn Leu Leu Asn Ala Asn Val Asp Ser Gly Leu Met His Ser He Gly 820 825 830 Leu Gly Tyr His Lys Asp Leu Gin Thr Arg Ala Thr Phe Met Glu Val 835 840 845 Leu Thr Lys He Leu Gin Gin Gly Thr Glu Phe Asp Thr Leu Ala Glu 850 855 860 Thr Val Leu Ala Asp Arg Phe Glu Arg Leu Val Glu Leu Val Thr Met 865 870 875 880 Met Gly Asp Gin Gly Glu Leu Pro He Ala Met Ala Leu Ala Asn Val 885 890 895 Val Pro Cys Ser Gin Trp Asp Glu Leu Ala Arg Val Leu Val Thr Leu 900 905 910 Phe Asp Ser Arg His Leu Leu Tyr Gin Leu Leu Trp Asn Met Phe Ser 915 920 925 Lys Glu Val Glu Leu Ala Asp Ser Met Gin Thr Leu Phe Arg Gly Asn 930 935 940 Ser Leu Ala Ser Lys He Met Thr Phe Cys Phe Lys Val Tyr Gly Ala 945 950 955 960 Thr Tyr Leu Gin Lys Leu Leu Asp Pro Leu Leu Arg He Val He Thr 965 970 975 Ser Ser Asp Trp Gin His Val Ser Phe Glu Val Asp Pro Thr Arg Leu 980 985 990 Glu Pro Ser Glu Ser Leu Glu Glu Asn Gin Arg Asn Leu Leu Gin Met 995 1000 1005 Thr Glu Lys Phe Phe His Ala He He Ser Ser Ser Ser Glu Phe Pro 1010 1015 1020 Pro Gin Leu Arg Ser Val Cys His Cys Leu Tyr Gin Val Val Ser Gin 1025 1030 1035 1040 Arg Phe Pro Gin Asn Ser He Gly Ala Val Gly Ser Ala Met Phe Leu 1045 1050 1055 Arg Phe He Asn Pro Ala He Val Ser Pro Tyr Glu Ala Gly He Leu 1060 1065 1070 Asp Lys Lys Pro Pro Pro Arg He Glu Arg Gly Leu Lys Leu Met Ser 1075 1080 1085 Lys He Leu Gin Ser He Ala Asn His Val Leu Leu Thr Lys Glu Glu 1090 1095 1100 His Met Arg Pro Phe Asn Asp Phe Val Lys Ser Asn Phe Asp Ala Ala 1105 1110 1115 1120 Arg Arg Phe Phe Leu Asp He Ala Ser Asp Cys Pro Thr Ser Asp Ala 1125 1130 1135 Val Asn His Ser Leu Ser Phe He Ser Asp Gly Asn Val Leu Ala Leu 1140 1145 1150 His Arg Leu Leu Trp Asn Asn Gin Glu Lys He Gly Gin Tyr Leu Ser 1155 1160 1165 Ser Asn Arg Asp His Lys Ala Val Gly Arg Arg Pro Phe Asp Lys Met 1170 1175 1180 Ala Thr Leu Leu Ala Tyr Leu Gly Pro Pro Glu His Lys Pro Val Ala 1185 1190 1195 1200 Asp Thr His Trp Ser Ser Leu Asn Leu Thr Ser Ser Lys Phe Glu Glu 1205 1210 1215 Phe Met Thr Arg His His Gin Val His Glu Lys Glu Glu Phe Lys Ala 1220 1225 1230 Leu Lys Thr Leu Ser He Phe Tyr Gin Ala Gly Thr Ser Lys Ala Gly 1235 1240 1245 Asn Pro He Phe Tyr Tyr Val Ala Arg Arg Phe Lys Thr Gly Gin He 1250 1255 1260 Asn Gly Asp Leu Leu He Tyr His Val Leu Leu Thr Leu Lys Pro Tyr 1265 1270 1275 1280 Tyr Ala Lys Pro Tyr Glu He Val Val Asp Leu Thr His Thr Gly Pro 1285 1290 1295 Ser Asn Arg Phe Lys Thr Asp Phe Leu Ser Lys Trp Phe Val Val Phe 1300 1305 1310 Pro Gly Phe Ala Tyr Asp Asn Val Ser Ala Val Tyr He Tyr Asn Cys 1315 1320 1325 Asn Ser Trp Val Arg Glu Tyr Thr Lys Tyr His Glu Arg Leu Leu Thr 1330 1335 1340 Gly Leu Lys Gly Ser Lys Arg Leu Val Phe He Asp Cys Pro Gly Lys 1345 1350 1355 1360 Leu Ala Glu His He Glu His Glu Gin Gin Lys Leu Pro Ala Ala Thr 1365 1370 1375 Leu Ala Leu Glu Glu Asp Leu Lys Val Phe His Asn Ala Leu Lys Leu 1380 1385 1390 Ala His Lys Asp Thr Lys Val Ser He Lys Val Gly Ser Thr Ala Val 1395 1400 1405 Gin Val Thr Ser Ala Glu Arg Thr Lys Val Leu Gly Gin Ser Val Phe 1410 1415 1420 Leu Asn Asp He Tyr Tyr Ala Ser Glu He Glu Glu He Cys Leu Val 1425 1430 1435 1440 Asp Glu Asn Gin Phe Thr Leu Thr He Ala Asn Gin Gly Thr Pro Leu 1445 1450 1455 Thr Phe Met His Gin Glu Cys Glu Ala He Val Gin Ser He He His 1460 1465 1470 He Arg Thr Arg Trp Glu Leu Ser Gin Pro Asp Ser He Pro Gin His 1475 1480 1485 Thr Lys He Arg Pro Lys Asp Val Pro Gly Thr Leu Leu Asn He Ala 1490 1495 1500 Leu Leu Asn Leu Gly Ser Ser Asp Pro Ser Leu Arg Ser Ala Ala Tyr 1505 1510 1515 1520 Asn Leu Leu Cys Ala Leu Thr Cys Thr Phe Asn Leu Lys He Glu Gly 1525 1530 1535 Gin Leu Leu Glu Thr Ser Gly Leu Cys He Pro Ala Asn Asn Thr Leu 1540 1545 1550 Phe He Val Ser He Ser Lys Thr Leu Ala Ala Asn Glu Pro His Leu 1555 1560 1565 Thr Leu Glu Phe Leu Glu Glu Cys He Ser Gly Phe Ser Lys Ser Ser 1570 1575 1580 He Glu Leu Lys His Leu Cys Leu Glu Tyr Met Thr Pro Trp Leu Ser 1585 1590 1595 1600 Asn Leu Val Arg Phe Cys Lys His Asn Asp Asp Ala Lys Arg Gin Arg 1605 1610 1615 Val Thr Ala He Leu Asp Lys Leu He Thr Met Thr He Asn Glu Lys 1620 1625 1630 Gin Met Tyr Pro Ser He Gin Ala Lys He Trp Gly Ser Leu Gly Gin 1635 1640 1645 He Thr Asp Leu Leu Asp Val Val Leu Asp Ser Phe He Lys Thr Ser 1650 1655 1660 Ala Thr Gly Gly Leu Gly Ser He Lys Ala Glu Val Met Ala Asp Thr 1665 1670 1675 1680 Ala Val Ala Leu Ala Ser Gly Asn Val Lys Leu Val Ser Ser Lys Val 1685 1690 1695 He Gly Arg Met Cys Lys He He Asp Lys Thr Cys Leu Ser Pro Thr 1700 1705 1710 Pro Thr Leu Glu Gin His Leu Met Trp Asp Asp He Ala He Leu Ala 1715 1720 1725 Arg Tyr Met Leu Met Leu Ser Phe Asn Asn Ser Leu Asp Val Ala Ala 1730 1735 1740 His Leu Pro Tyr Leu Phe His Val Val Thr Phe Leu Val Ala Thr Gly 1745 1750 1755 1760 Pro Leu Ser Leu Arg Ala Ser Thr His Gly Leu Val He Asn He He 1765 1770 1775 His Ser Leu Cys Thr Cys Ser Gin Leu His Phe Ser Glu Glu Thr Lys 1780 1785 1790 Gin Val Leu Arg Leu Ser Leu Thr Glu Phe Ser Leu Pro Lys Phe Tyr 1795 1800 1805 Leu Leu Phe Gly He Ser Lys Val Lys Ser Ala Ala Val He Ala Phe 1810 1815 1820 Arg Ser Ser Tyr Arg Asp Arg Ser Phe Ser Pro Gly Ser Tyr Glu Arg 1825 1830 1835 1840 Glu Thr Phe Ala Leu Thr Ser Leu Glu Thr Val Thr Glu Ala Leu Leu 1845 1850 1855 Glu He Met Glu Ala Cys Met Arg Asp He Pro Thr Cys Lys Trp Leu 1860 1865 1870 Asp Gin Trp Thr Glu Leu Ala Gin Arg Phe Ala Phe Gin Tyr Asn Pro 1875 1880 1885 Ser Leu Gin Pro Arg Ala Leu Val Val Phe Gly Cys He Ser Lys Arg 1890 1895 1900 Val Ser His Gly Gin He Lys Gin He He Arg He Leu Ser Lys Ala 1905 1910 1915 1920 Leu Glu Ser Cys Leu Lys Gly Pro Asp Thr Tyr Asn Ser Gin Val Leu 1925 1930 1935 He Glu Ala Thr Val He Ala Leu Thr Lys Leu Gin Pro Leu Leu Asn 1940 1945 1950 Lys Asp Ser Pro Leu His Lys Ala Leu Phe Trp Val Ala Val Ala Val 1955 1960 1965 Leu Gin Leu Asp Glu Val Asn Leu Tyr Ser Ala Gly Thr Ala Leu Leu 1970 1975 1980 Glu Gin Asn Leu His Thr Leu Asp Ser Leu Arg He Phe Asn Asp Lys 1985 1990 1995 2000 Ser Pro Glu Glu Val Phe Met Ala He Arg Asn Pro Leu Glu Trp His 2005 2010 2015 Cys Lys Gin Met Asp His Phe Val Gly Leu Asn Phe Asn Ser Asn Phe 2020 2025 2030 Asn Phe Ala Leu Val Gly His Leu Leu Lys Gly Tyr Arg His Pro Ser 2035 2040 2045 Pro Ala He Val Ala Arg Thr Val Arg He Leu His Thr Leu Leu Thr 2050 2055 2060 Leu Val Asn Lys His Arg Asn Cys Asp Lys Phe Glu Val Asn Thr Gin 2065 2070 2075 2080 Ser Val Ala Tyr Leu Ala Ala Leu Leu Thr Val Ser Glu Glu Val Arg 2085 2090 2095 Ser Arg Cys Ser Leu Lys His Arg Lys Ser Leu Leu Leu Thr Asp He 2100 2105 2110 Ser Met Glu Asn Val Pro Met Asp Thr Tyr Pro He His His Gly Asp 2115 2120 2125 Pro Ser Tyr Arg Thr Leu Lys Glu Thr Gin Pro Trp Ser Ser Pro Lys 2130 2135 2140 Gly Ser Glu Gly Tyr Leu Ala Ala Thr Tyr Pro Thr Val Gly Gin Thr 2145 2150 2155 2160 Ser Pro Arg Ala Arg Lys Ser Met Ser Leu Asp Met Gly Gin Pro Ser 2165 2170 2175 Gin Ala Asn Thr Lys Lys Leu Leu Gly Thr Arg Lys Ser Phe Asp His 2180 2185 2190 Leu He Ser Asp Thr Lys Ala Pro Lys Arg Gin Glu Met Glu Ser Gly 2195 2200 2205 He Thr Thr Pro Pro Lys Met Arg Arg Val Ala Glu Thr Asp Tyr Glu 2210 2215 2220 Met Glu Thr Gin Arg He Ser Ser Ser Gin Gin His Pro His Leu Arg 2225 2230 2235 2240 Lys Val Ser Val Ser Glu Ser Asn Val Leu Leu Asp Glu Glu Val Leu 2245 2250 2255 Thr Asp Pro Lys He Gin Ala Leu Leu Leu Thr Val Leu Ala Thr Leu 2260 2265 2270 Val Lys Tyr Thr Thr Asp Glu Phe Asp Gin Arg He Leu Tyr Glu Tyr 2275 2280 2285 Leu Ala Glu Ala Ser Val Val Phe Pro Lys Val Phe Pro Val Val His 2290 2295 2300 Asn Leu Leu Asp Ser Lys He Asn Thr Leu Leu Ser Leu Cys Gin Asp 2305 2310 2315 2320 Pro Asn Leu Leu Asn Pro He His Gly He Val Gin Ser Val Val Tyr 2325 2330 2335 His Glu Glu Ser Pro Pro Gin Tyr Gin Thr Ser Tyr Leu Gin Ser Phe 2340 2345 2350 Gly Phe Asn Gly Leu Trp Arg Phe Ala Gly Pro Phe Ser Lys Gin Thr 2355 2360 2365 Gin He Pro Asp Tyr Ala Glu Leu He Val Lys Phe Leu Asp Ala Leu 2370 2375 2380 He Asp Thr Tyr Leu Pro Gly He Asp Glu Glu Thr Ser Glu Glu Ser 2385 2390 2395 2400 Leu Leu Thr Pro Thr Ser Pro Tyr Pro Pro Ala Leu Gin Ser Gin Leu 2405 2410 2415 Ser He Thr Ala Asn Leu Asn Leu Ser Asn Ser Met Thr Ser Leu Ala 2420 2425 2430 Thr Ser Gin His Ser Pro Gly He Asp Lys Glu Asn Val Glu Leu Ser 2435 2440 2445 Pro Thr Thr Gly His Cys Asn Ser Gly Arg Thr Arg His Gly Ser Ala 2450 2455 2460 Ser Gin Val Gin Lys Gin Arg Ser Ala Gly Ser Phe Lys Arg Asn Ser 2465 2470 2475 2480 He Lys Lys He Val 2485 (2) INFORMATION FOR SEQ ID N0:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2485 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asn Trp Glu Asp Asn Ser Val He Phe Leu Leu Val Gin Ser Met Val 1 5 10 15 Val Asp Leu Lys Asn Leu Leu Phe Asn Pro Ser Lys Pro Phe Ser Arg 20 25 30 Gly Ser Gin Pro Ala Asp Val Asp Leu Met He Asp Cys Leu Val Ser 35 40 45 Cys Phe Arg He Ser Pro His Asn Asn Gin His Phe Lys He Cys Leu 50 55 60 Ala Gin Asn Ser Pro Ser Thr Phe His Tyr Val Leu Val Asn Ser Leu 65 70 75 80 His Arg He He Thr Asn Ser Ala Leu Asp Trp Trp Pro Lys He Asp 85 90 95 Ala Val Tyr Cys His Ser Val Glu Leu Arg Asn Met Phe Gly Glu Thr 100 105 110 Leu His Lys Ala Val Gin Gly Cys Gly Ala His Pro Ala He Arg Met 115 120 125 Ala Pro Ser Leu Thr Phe Lys Glu Lys Val Thr Ser Leu Lys Phe Lys 130 135 140 Glu Lys Pro Thr Asp Leu Glu Thr Arg Ser Tyr Lys Tyr Leu Leu Leu 145 150 155 160 Ser He Val Lys Leu He His Ala Asp Pro Lys Leu Leu Leu Cys Asn 165 170 175 Pro Arg Lys Gin Gly Pro Glu Thr Gin Gly Ser Thr Ala Glu Leu He 180 185 190 Thr Gly Leu Val Gin Leu Val Pro Gin Ser His Met Pro Glu He Ala 195 200 205 Gin Glu Ala Met Glu Ala Leu Leu Val Leu His Gin Leu Asp Ser He 210 215 220 Asp Leu Trp Asn Pro Asp Ala Pro Val Glu Thr Phe Trp Glu He Ser 225 230 235 240 Ser Gin Met Leu Phe Tyr He Cys Lys Lys Leu Thr Ser His Gin Met 245 250 255 Leu Ser Ser Thr Glu He Leu Lys Trp Leu Arg Glu He Leu He Cys 260 265 270 Arg Asn Lys Phe Leu Leu Lys Asn Lys Gin Ala Asp Arg Ser Ser Cys 275 280 285 His Phe Leu Leu Phe Tyr Gly Val Gly Cys Asp He Pro Ser Ser Gly 290 295 300 Asn Thr Ser Gin Met Ser Met Asp His Glu Glu Leu Leu Arg Thr Pro 305 310 315 320 Gly Ala Ser Leu Arg Lys Gly Lys Gly Asn Ser Ser Met Asp Ser Ala 325 330 335 Ala Gly Cys Ser Gly Thr Pro Pro He Cys Arg Gin Ala Gin Thr Lys 340 345 350 Leu Glu Val Ala Leu Tyr Met Phe Leu Trp Asn Pro Asp Thr Glu Ala 355 360 365 Val Leu Val Ala Met Ser Cys Phe Arg His Leu Cys Glu Glu Ala Asp 370 375 380 He Arg Cys Gly Val Asp Glu Val Ser Val His Asn Leu Leu Pro Asn 385 390 395 400 Tyr Asn Thr Phe Met Glu Phe Ala Ser Val Ser Asn Met Met Ser Thr 405 410 415 Gly Arg Ala Ala Leu Gin Lys Arg Val Met Ala Leu Leu Arg Arg He 420 425 430 Glu His Pro Thr Ala Gly Asn Thr Glu Ala Trp Glu Asp Thr His Ala 435 440 445 Lys Trp Glu Gin Ala Thr Lys Leu He Leu Asn Tyr Pro Lys Ala Lys 450 455 460 Met Glu Asp Gly Gin Ala Ala Glu Ser Leu His Lys Thr He Val Lys 465 470 475 480 Arg Arg Met Ser His Val Ser Gly Gly Gly Ser He Asp Leu Ser Asp 485 490 495 Thr Asp Ser Leu Gin Glu Trp He Asn Met Thr Gly Phe Leu Cys Ala 500 505 510 Leu Gly Gly Val Cys Leu Gin Gin Arg Ser Asn Ser Gly Leu Ala Thr 515 520 525 Tyr Ser Pro Pro Met Gly Pro Val Ser Glu Arg Lys Gly Ser Met He 530 535 540 Ser Val Met Ser Ser Glu Gly Asn Ala Asp Thr Pro Val Ser Lys Phe 545 550 555 560 Met Asp Arg Leu Leu Ser Leu Met Val Cys Asn His Glu Lys Val Gly 565 570 575 Leu Gin He Arg Thr Asn Val Lys Asp Leu Val Gly Leu Glu Leu Ser 580 585 590 Pro Ala Leu Tyr Pro Met Leu Phe Asn Lys Leu Lys Asn Thr He Ser 595 600 605 Lys Phe Phe Asp Ser Gin Gly Gin Val Leu Leu Thr Asp Thr Asn Thr 610 615 620 Gin Phe Val Glu Gin Thr He Ala He Met Lys Asn Leu Leu Asp Asn 625 630 635 640 His Thr Glu Gly Ser Ser Glu His Leu Gly Gin Ala Ser He Glu Thr 645 650 655 Met Met Leu Asn Leu Val Arg Tyr Val Arg Val Leu Gly Asn Met Val 660 665 670 His Ala He Gin He Lys Thr Lys Leu Cys Gin Leu Val Glu Val Met 675 680 685 Met Ala Arg Arg Asp Asp Leu Ser Phe Cys Gin Glu Met Lys Phe Arg 690 695 700 Asn Lys Met Val Glu Tyr Leu Thr Asp Trp Val Met Gly Thr Ser Asn 705 710 715 720 Gin Ala Ala Asp Asp Asp Val Lys Cys Leu Thr Arg Asp Leu Asp Gin 725 730 735 Ala Ser Met Glu Ala Val Val Ser Leu Leu Ala Gly Leu Pro Leu Gin 740 745 750 Pro Glu Glu Gly Asp Gly Val Glu Leu Met Glu Ala Lys Ser Gin Leu 755 760 765 Phe Leu Lys Tyr Phe Thr Leu Phe Met Asn Leu Leu Asn Asp Cys Ser 770 775 780 Glu Val Glu Asp Glu Ser Ala Gin Thr Gly Gly Arg Lys Arg Gly Met 785 790 795 800 Ser Arg Arg Leu Ala Ser Leu Arg His Cys Thr Val Leu Ala Met Ser 805 810 815 Asn Leu Leu Asn Ala Asn Val Asp Ser Gly Leu Met His Ser He Gly 820 825 830 Leu Gly Tyr His Lys Asp Leu Gin Thr Arg Ala Thr Phe Met Glu Val 835 840 845 Leu Thr Lys He Leu Gin Gin Gly Thr Glu Phe Asp Thr Leu Ala Glu 850 855 860 Thr Val Leu Ala Asp Arg Phe Glu Arg Leu Val Glu Leu Val Thr Met 865 870 875 880 Met Gly Asp Gin Gly Glu Leu Pro He Ala Met Ala Leu Ala Asn Val 885 890 895 Val Pro Cys Ser Gin Trp Asp Glu Leu Ala Arg Val Leu Val Thr Leu 900 905 910 Phe Asp Ser Arg His Leu Leu Tyr Gin Leu Leu Trp Asn Met Phe Ser 915 920 925 Lys Glu Val Glu Leu Ala Asp Ser Met Gin Thr Leu Phe Arg Gly Asn 930 935 940 Ser Leu Ala Ser Lys He Met Thr Phe Cys Phe Lys Val Tyr Gly Ala 945 950 955 960 Thr Tyr Leu Gin Lys Leu Leu Asp Pro Leu Leu Arg He Val He Thr 965 970 975 Ser Ser Asp Trp Gin His Val Ser Phe Glu Val Asp Pro Thr Arg Leu 980 985 990 Glu Pro Ser Glu Ser Leu Glu Glu Asn Gin Arg Asn Leu Leu Gin Met 995 1000 1005 Thr Glu Lys Phe Phe His Ala He He Ser Ser Ser Ser Glu Phe Pro 1010 1015 1020 Pro Gin Leu Arg Ser Val Cys His Cys Leu Tyr Gin Val Val Ser Gin 1025 1030 1035 1040 Arg Phe Pro Gin Asn Ser He Gly Ala Val Gly Ser Ala Met Phe Leu 1045 1050 1055 Arg Phe He Asn Pro Ala He Val Ser Pro Tyr Glu Ala Gly He Leu 1060 1065 1070 Asp Lys Lys Pro Pro Pro Arg He Glu Arg Gly Leu Lys Leu Met Ser 1075 1080 1085 Lys He Leu Gin Ser He Ala Asn His Val Leu Phe Thr Arg Glu Glu 1090 1095 1100 His Met Arg Pro Phe Asn Asp Phe Val Lys Ser Asn Phe Asp Ala Ala 1105 1110 1115 1120 Arg Arg Phe Phe Leu Asp He Ala Ser Asp Cys Pro Thr Ser Asp Ala 1125 1130 1135 Val Asn His Ser Leu Ser Phe He Ser Asp Gly Asn Val Leu Ala Leu 1140 1145 1150 His Arg Leu Leu Trp Asn Asn Gin Glu Lys He Gly Gin Tyr Leu Ser 1155 1160 1165 Ser Asn ,Arg Asp His Lys Ala Val Gly Arg Arg Pro Phe Asp Lys Met 1170 1175 1180 Ala Thr Leu Leu Ala Tyr Leu Gly Pro Pro Glu His Lys Pro Val Ala 1185 1190 1195 1200 Asp Thr His Trp Ser Ser Leu Asn Leu Thr Ser Ser Lys Phe Glu Glu 1205 1210 1215 Phe Met Thr Arg His His Gin Val His Glu Lys Glu Glu Phe Lys Ala 1220 1225 1230 Leu Lys Thr Leu Ser He Phe Tyr Gin Ala Gly Thr Ser Lys Ala Gly 1235 1240 1245 Asn Pro He Phe Tyr Tyr Val Ala Arg Arg Phe Lys Thr Gly Gin He 1250 1255 1260 Asn Gly Asp Leu Leu He Tyr His Val Leu Leu Thr Leu Lys Pro Tyr 1265 1270 1275 1280 Tyr Ala Lys Pro Tyr Glu He Val Val Asp Leu Thr His Thr Gly Pro 1285 1290 1295 Ser Asn Arg Phe Lys Thr Asp Phe Leu Ser Lys Trp Phe Val Val Phe 1300 1305 1310 Pro Gly Phe Ala Tyr Asp Asn Val Ser Ala Val Tyr He Tyr Asn Cys 1315 1320 1325 Asn Ser Trp Val Arg Glu Tyr Thr Lys Tyr His Glu Arg Leu Leu Thr 1330 1335 1340 Gly Leu Lys Gly Ser Lys Arg Leu Val Phe He Asp Cys Pro Gly Lys 1345 1350 1355 1360 Leu Ala Glu His He Glu His Glu Gin Gin Lys Leu Pro Ala Ala Thr 1365 1370 1375 Leu Ala Leu Glu Glu Asp Leu Lys Val Phe His Asn Ala Leu Lys Leu 1380 1385 1390 Ala His Lys Asp Thr Lys Val Ser He Lys Val Gly Ser Thr Ala Val 1395 1400 1405 Gin Val Thr Ser Ala Glu Arg Thr Lys Val Leu Gly Gin Ser Val Phe 1410 1415 1420 Leu Asn Asp He Tyr Tyr Ala Ser Glu He Glu Glu He Cys Leu Val 1425 1430 1435 1440 Asp Glu Asn Gin Phe Thr Leu Thr He Ala Asn Gin Gly Thr Pro Leu 1445 1450 1455 Thr Phe Met His Gin Glu Cys Glu Ala He Val Gin Ser He He His 1460 1465 1470 He Arg Thr Arg Trp Glu Leu Ser Gin Pro Asp Ser He Pro Gin His 1475 1480 1485 Thr Lys He Arg Pro Lys Asp Val Pro Gly Thr Leu Leu Asn He Ala 1490 1495 1500 Leu Leu Asn Leu Gly Ser Ser Asp Pro Ser Leu Arg Ser Ala Ala Tyr 1505 1510 1515 1520 Asn Leu Leu Cys Ala Leu Thr Cys Thr Phe Asn Leu Lys He Glu Gly 1525 1530 1535 Gin Leu Leu Glu Thr Ser Gly Leu Cys He Pro Ala Asn Asn Thr Leu 1540 1545 1550 Phe He Val Ser He Ser Lys Thr Leu Ala Ala Asn Glu Pro His Leu 1555 1560 1565 Thr Leu Glu Phe Leu Glu Glu Cys He Ser Gly Phe Ser Lys Ser Ser 1570 1575 1580 He Glu Leu Lys His Leu Cys Leu Glu Tyr Met Thr Pro Trp Leu Ser 1585 1590 1595 1600 Asn Leu Val Arg Phe Cys Lys His Asn Asp Asp Ala Lys Arg Gin Arg 1605 1610 1615 Val Thr Ala He Leu Asp Lys Leu He Thr Met Thr He Asn Glu Lys 1620 1625 1630 Gin Met Tyr Pro Ser He Gin Ala Lys He Trp Gly Ser Leu Gly Gin 1635 1640 1645 He Thr Asp Leu Leu Asp Val Val Leu Asp Ser Phe He Lys Thr Ser 1650 1655 1660 Ala Thr Gly Gly Leu Gly Ser He Lys Ala Glu Val Met Ala Asp Thr 1665 1670 1675 1680 Ala Val Ala Leu Ala Ser Gly Asn Val Lys Leu Val Ser Ser Lys Val 1685 1690 1695 He Gly Arg Met Cys Lys He He Asp Lys Thr Cys Leu Ser Pro Thr 1700 1705 1710 Pro Thr Leu Glu Gin His Leu Met Trp Asp Asp He Ala He Leu Ala 1715 1720 1725 Arg Tyr Met Leu Met Leu Ser Phe Asn Asn Ser Leu Asp Val Ala Ala 1730 1735 1740 His Leu Pro Tyr Leu Phe His Val Val Thr Phe Leu Val Ala Thr Gly 1745 1750 1755 1760 Pro Leu Ser Leu Arg Ala Ser Thr His Gly Leu Val He Asn He He 1765 1770 1775 His Ser Leu Cys Thr Cys Ser Gin Leu His Phe Ser Glu Glu Thr Lys 1780 1785 1790 Gin Val Leu Arg Leu Ser Leu Thr Glu Phe Ser Leu Pro Lys Phe Tyr 1795 1800 1805 Leu Leu Phe Gly He Ser Lys Val Lys Ser Ala Ala Val He Ala Phe 1810 1815 1820 Arg Ser Ser Tyr Arg Asp Arg Ser Phe Ser Pro Gly Ser Tyr Glu Arg 1825 1830 1835 1840 Glu Thr Phe Ala Leu Thr Ser Leu Glu Thr Val Thr Glu Ala Leu Leu 1845 1850 1855 Glu He Met Glu Ala Cys Met Arg Asp He Pro Thr Cys Lys Trp Leu 1860 1865 1870 Asp Gin Trp Thr Glu Leu Ala Gin Arg Phe Ala Phe Gin Tyr Asn Pro 1875 1880 1885 Ser Leu Gin Pro Arg Ala Leu Val Val Phe Gly Cys He Ser Lys Arg 1890 1895 1900 Val Ser His Gly Gin He Lys Gin He He Arg He Leu Ser Lys Ala 1905 1910 1915 1920 Leu Glu Ser Cys Leu Lys Gly Pro Asp Thr Tyr Asn Ser Gin Val Leu 1925 1930 1935 He Glu Ala Thr Val He Ala Leu Thr Lys Leu Gin Pro Leu Leu Asn 1940 1945 1950 Lys Asp Ser Pro Leu His Lys Ala Leu Phe Trp Val Ala Val Ala Val 1955 1960 1965 Leu Gin Leu Asp Glu Val Asn Leu Tyr Ser Ala Gly Thr Ala Leu Leu 1970 1975 1980 Glu Gin Asn Leu His Thr Leu Asp Ser Leu Arg He Phe Asn Asp Lys 1985 1990 1995 2000 Ser Pro Glu Glu Val Phe Met Ala He Arg Asn Pro Leu Glu Trp His 2005 2010 2015 Cys Lys Gin Met Asp His Phe Val Gly Leu Asn Phe Asn Ser Asn Phe 2020 2025 2030 Asn Phe Ala Leu Val Gly His Leu Leu Lys Gly Tyr Arg His Pro Ser 2035 2040 2045 Pro Ala He Val Ala Arg Thr Val Arg He Leu His Thr Leu Leu Thr 2050 2055 2060 Leu Val Asn Lys His Arg Asn Cys Asp Lys Phe Glu Val Asn Thr Gin 2065 2070 2075 2080 Ser Val Ala Tyr Leu Ala Ala Leu Leu Thr Val Ser Glu Glu Val Arg 2085 2090 2095 Ser Arg Cys Ser Leu Lys His Arg Lys Ser Leu Leu Leu Thr Asp He 2100 2105 2110 Ser Met Glu Asn Val Pro Met Asp Thr Tyr Pro He His His Gly Asp 2115 2120 2125 Pro Ser Tyr Arg Thr Leu Lys Glu Thr Gin Pro Trp Ser Ser Pro Lys 2130 2135 2140 Gly Ser Glu Gly Tyr Leu Ala Ala Thr Tyr Pro Thr Val Gly Gin Thr 2145 2150 2155 2160 Ser Pro Arg Ala Arg Lys Ser Met Ser Leu Asp Met Gly Gin Pro Ser 2165 2170 2175 Gin Ala Asn Thr Lys Lys Leu Leu Gly Thr Arg Lys Ser Phe Asp His 2180 2185 2190 Leu He Ser Asp Thr Lys Ala Pro Lys Arg Gin Glu Met Glu Ser Gly 2195 2200 2205 He Thr Thr Pro Pro Lys Met Arg Arg Val Ala Glu Thr Asp Tyr Glu 2210 2215 2220 Met Glu Thr Gin Arg He Ser Ser Ser Gin Gin His Pro His Leu Arg 2225 2230 2235 2240 Lys Val Ser Val Ser Glu Ser Asn Val Leu Leu Asp Glu Glu Val Leu 2245 2250 2255 Thr Asp Pro Lys He Gin Ala Leu Leu Leu Thr Val Leu Ala Thr Leu 2260 2265 2270 Val Lys Tyr Thr Thr Asp Glu Phe Asp Gin Arg He Leu Tyr Glu Tyr 2275 2280 2285 Leu Ala Glu Ala Ser Val Val Phe Pro Lys Val Phe Pro Val Val His 2290 2295 2300 Asn Leu Leu Asp Ser Lys He Asn Thr Leu Leu Ser Leu Cys Gin Asp 2305 2310 2315 2320 Pro Asn Leu Leu Asn Pro He His Gly He Val Gin Ser Val Val Tyr 2325 2330 2335 His Glu Glu Ser Pro Pro Gin Tyr Gin Thr Ser Tyr Leu Gin Ser Phe 2340 2345 2350 Gly Phe Asn Gly Leu Trp Arg Phe Ala Gly Pro Phe Ser Lys Gin Thr 2355 2360 2365 Gin He Pro Asp Tyr Ala Glu Leu He Val Lys Phe Leu Asp Ala Leu 2370 2375 2380 He Asp Thr Tyr Leu Pro Gly He Asp Glu Glu Thr Ser Glu Glu Ser 2385 2390 2395 2400 Leu Leu Thr Pro Thr Ser Pro Tyr Pro Pro Ala Leu Gin Ser Gin Leu 2405 2410 2415 Ser He Thr Ala Asn Leu Asn Leu Ser Asn Ser Met Thr Ser Leu Ala 2420 2425 2430 Thr Ser Gin His Ser Pro Ala Ser Leu Pro Cys Ser Asn Ser Ala Val 2435 2440 2445 Phe Met Gin Leu Phe Pro His Gin Gly He Asp Lys Glu Asn Val Glu 2450 2455 2460 Leu Ser Pro Thr Thr Gly His Cys Asn Ser Gly Arg Thr Arg His Gly 2465 2470 2475 2480 Ser Ala Ser Gin Val 2485 INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2938 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Leu Leu Cys Lys He Ser Lys Leu Lys Phe Asn Thr Arg Thr Leu 1 5 10 15 Lys Val Leu Gin Asn Met Ser His His Leu Ser Gly Ser Ala Thr He 20 25 30 Ser Lys Ser Ser He Leu Pro Asp Ser Gin Glu Phe Leu Gin Lys Arg 35 40 45 Asn Tyr Pro Ala Tyr Thr Glu Lys He Asp Leu Thr He Asp Tyr He 50 55 60 Gin Arg Phe He Ser Ala Ser Asn His Val Glu Phe Thr Lys Cys Val 65 70 75 80 Lys Thr Lys Val Val Ala Pro Leu Leu He Ser His Thr Ser Thr Glu 85 90 95 Leu Gly Val Val Asn His Leu Asp Leu Phe Gly Cys Glu Tyr Leu Thr 100 105 110 Asp Lys Asn Leu Leu Ala Tyr Leu Asp He Leu Gin His Leu Ser Ser 115 120 125 Tyr Met Lys Arg Thr He Phe His Ser Leu Leu Leu Tyr Tyr Ala Ser 130 135 140 Lys Ala Phe Leu Phe Trp He Met Ala Arg Pro Lys Glu Tyr Val Lys 145 150 155 160 He Tyr Asn Asn Leu He Ser Ser Asp Tyr Asn Ser Pro Ser Ser Ser 165 170 175 Ser Asp Asn Gly Gly Ser Asn Asn Ser Asp Lys Thr Ser He Ser Gin 180 185 190 Leu Val Ser Leu Leu Phe Asp Asp Val Tyr Ser Thr Phe Ser Gly Ser 195 200 205 Ser Leu Leu Thr Asn Val Asn Asn Asp His His Tyr His Leu His His 210 215 220 Ser Ser Ser Ser Ser Lys Thr Thr Asn Thr Asn Ser Pro Asn Ser He 225 230 235 240 Ser Lys Thr Ser He Lys Gin Ser Ser Val Asn Ala Ser Gly Asn Val 245 250 255 Ser Pro Ser Gin Phe Ser Thr Gly Asn Asp Ala Ser Pro Thr Ser Pro 260 265 270 Met Ala Ser Leu Ser Ser Pro Leu Asn Thr Asn He Leu Gly Tyr Pro 275 280 285 Leu Ser Pro He Thr Ser Thr Leu Gly Gin Ala Asn Thr Ser Thr Ser 290 295 300 Thr Thr Ala Ala Thr Thr Lys Thr Asp Ala Asp Thr Pro Ser Thr Met 305 310 315 320 Asn Thr Asn Asn Asn Asn Asn Asn Asn Asn Ser Ala Asn Leu Asn Asn 325 330 335 He Pro Gin Arg He Phe Ser Leu Asp Asp He Ser Ser Phe Asn Ser 340 345 350 Ser Arg Lys Ser Leu Asn Leu Asp Asp Ser Asn Ser Leu Phe Leu Trp 355 360 365 Asp Thr Ser Gin His Ser Asn Ala Ser Met Thr Asn Thr Asn Met His 370 375 380 Ala Gly Val Asn Asn Ser Gin Ser Gin Asn Asp Gin Ser Ser Leu Asn 385 390 395 400 Tyr Met Glu Asn He Met Glu Leu Tyr Ser Asn Tyr Thr Gly Ser Glu 405 410 415 Leu Ser Ser His Thr Ala He Leu Arg Phe Leu Val Val Leu Thr Leu 420 425 430 Leu Asp Ser Glu Val Tyr Asp Glu Met Asn Ser Asn Ser Tyr Arg Lys 435 440 445 He Ser Glu Pro He Met Asn He Asn Pro Lys Asp Ser Asn Thr Ser 450 455 460 Ser Trp Gly Ser Ala Ser Lys Asn Pro Ser He Arg His Leu Thr His 465 470 475 480 Gly Leu Lys Lys Leu Thr Leu Gin Gin Gly Arg Lys Arg Asn Val Lys 485 490 495 Phe Leu Thr Tyr Leu He Arg Asn Leu Asn Gly Gly Gin Phe Val Ser 500 505 510 Asp Val Ser Leu He Asp Ser He Arg Ser He Leu Phe Leu Met Thr 515 520 525 Met Thr Ser Ser He Ser Gin He Asp Ser Asn He Ala Ser Val He 530 535 540 Phe Ser Lys Arg Phe Tyr Asn Leu Leu Gly Gin Asn Leu Glu Val Gly 545 550 555 560 Thr Asn Trp Asn Ser Ala Thr Ala Asn Thr Phe He Ser His Cys Val 565 570 575 Glu Arg Asn Pro Leu Thr His Arg Arg Leu Gin Leu Glu Phe Phe Ala 580 585 590 Ser Gly Leu Gin Leu Asp Ser Asp Leu Phe Leu Arg His Leu Gin Leu 595 600 605 Glu Lys Glu Leu Asn His He Asp Leu Pro Lys He Ser Leu Tyr Thr 610 615 620 Glu Gly Phe Arg Val Phe Phe His Leu Val Ser Thr Lys Lys Leu His 625 630 635 " 640 Glu Asp He Ala Glu Lys Thr Ser Ser Val Leu Lys Arg Leu Phe Cys 645 650 655 He He Ala Asp He Leu Leu Lys Ala Thr Pro Tyr Phe Asp Asp Asn 660 665 670 Val Thr Lys He He Ala Ser He Leu Asp Gly His He Leu Asp Gin 675 680 685 Phe Asp Ala Ala Arg Thr Leu Ser Asn Asp Asp His Val Ser Phe Asp 690 695 700 Ala Ala Thr Ser Val Tyr Thr Glu Pro Thr Glu He He His Asn Ser 705 710 715 720 Ser Asp Ala Ser Leu Val Ser Ser Leu Ser Gin Ser Pro Leu Ser He 725 730 735 Asn Ser Gly Ser Asn He Thr Asn Thr Arg Thr Trp Asp He Gin Ser 740 745 750 He Leu Pro Thr Leu Ser Asn Arg Ser Ser Ala Ser Asp Leu Ser Leu 755 760 765 Ser Asn He Leu Thr Asn Pro Leu Glu Ala Gin Gin Asn Asn Asn Ala 770 775 780 Asn Leu Leu Ala His Arg Leu Ser Gly Val Pro Thr Thr Lys Arg Tyr 785 790 795 800 Ala Ser Pro Asn Asp Ser Glu Arg Ser Arg Gin Ser Pro Tyr Ser Ser 805 810 815 Pro Pro Gin Leu Gin Gin Ser Asp Leu Pro Ser Pro Leu Ser Val Leu 820 825 830 Ser Ser Ser Ala Gly Phe Ser Ser Asn His Ser He Thr Ala Thr Pro 835 840 845 Thr He Leu Lys Asn He Lys Ser Pro Lys Pro Asn Lys Thr Lys Lys 850 855 860 He Ala Asp Asp Lys Gin Leu Lys Gin Pro Ser Tyr Ser Arg Val He 865 870 875 880 Leu Ser Asp Asn Asp Glu Ala Arg Lys He Met Met Asn He Phe Ser 885 890 895 He Phe Lys Arg Met Thr Asn Trp Phe He Arg Pro Asp Ala Asn Thr 900 905 910 Glu Phe Pro Lys Thr Phe Thr Asp He He Lys Pro Leu Phe Val Ser 915 920 925 He Leu Asp Ser Asn Gin Arg Leu Gin Val Thr Ala Arg Ala Phe He 930 935 940 Glu He Pro Leu Ser Tyr He Ala Thr Phe Glu Asp He Asp Asn Asp 945 950 955 960 Leu Asp Pro Arg Val Leu Asn Asp His Tyr Leu Leu Cys Thr Tyr Ala 965 970 975 Val Thr Leu Phe Ala Ser Ser Leu Phe Asp Leu Lys Leu Glu Asn Ala 980 985 990 Lys Arg Glu Met Leu Leu Asp He He Val Lys Phe Gin Arg Val Arg 995 1000 1005 Ser Tyr Leu Ser Asn Leu Ala Glu Lys His Asn Leu Val Gin Ala He 1010 1015 1020 He Thr Thr Glu Arg Leu Thr Leu Pro Leu Leu Val Gly Ala Val Gly 1025 1030 1035 1040 Ser Gly He Phe He Ser Leu Tyr Cys Ser Arg Gly Asn Thr Pro Arg 1045 1050 1055 Leu He Lys He Ser Cys Cys Glu Phe Leu Arg Ser Leu Arg Phe Tyr 1060 1065 1070 Gin Lys Tyr Val Gly Ala Leu Asp Gin Tyr Ser He Tyr Asn He Asp 1075 1080 1085 Phe He Asp Ala Met Ala Gin Asp Asn Phe Thr Ala Ser Gly Ser Val 1090 1095 1100 Ala Leu Gin Arg Arg Leu Arg Asn Asn He Leu Thr Tyr He Lys Gly 1105 1110 1115 1120 Ser Asp Ser He Leu Leu Asp Ser Met Asp Val He Tyr Lys Lys Trp 1125 1130 1135 Phe Tyr Phe Ser Cys Ser Lys Ser Val Thr Gin Glu Glu Leu Val Asp 1140 1145 1150 Phe Arg Ser Leu Ala Gly He Leu Ala Ser Met Ser Gly He Leu Ser 1155 1160 1165 Asp Met Gin Glu Leu Glu Lys Ser Lys Ser Ala Pro Asp Asn Glu Gly 1170 1175 1180 Asp Ser Leu Ser Phe Glu Ser Arg Asn Pro Ala Tyr Glu Val His Lys 1185 1190 1195 1200 Ser Leu Lys Leu Glu Leu Thr Lys Lys Met Asn Phe Phe He Ser Lys 1205 1210 1215 Gin Cys Gin Trp Leu Asn Asn Pro Asn Leu Leu Thr Arg Glu Asn Ser 1220 1225 1230 Arg Asp He Leu Ser He Glu Leu His Pro Leu Ser Phe Asn Leu Leu 1235 1240 1245 Phe Asn Asn Leu Gly Leu Lys He Asp Glu Leu Met Ser He Asp Leu 1250 1255 1260 Ser Lys Ser His Glu Asp Ser Ser Phe Val Leu Leu Glu Gin He He 1265 1270 1275 1280 He He He Arg Thr He Leu Lys Arg Asp Asp Asp Glu Lys He Met 1285 1290 1295 Leu Leu Phe Ser Thr Asp Leu Leu Asp Ala Val Asp Lys Leu He Glu 1300 1305 1310 He Val Glu Lys He Ser He Lys Ser Ser Lys Tyr Tyr Lys Gly He 1315 1320 1325 He Gin Met Ser Lys Met Phe Arg Ala Phe Glu His Ser Glu Lys Asn 1330 1335 1340 Leu Gly He Ser Asn His Phe His Leu Lys Asn Lys Trp Leu Lys Leu 1345 1350 1355 1360 Val He Gly Trp Phe Lys Leu Ser He Asn Lys Asp Tyr Asp Phe Glu 1365 1370 1375 Asn Leu Ser Arg Pro Leu Arg Glu Met Asp Leu Gin Lys Arg Asp Glu 1380 1385 1390 Asp Phe Leu Tyr He Asp Thr Ser He Glu Ser Ala Lys Ala Leu Ala 1395 1400 1405 Tyr Leu Thr His Asn Val Pro Leu Glu He Pro Pro Ser Ser Ser Lys 1410 1415 1420 Glu Asp Trp Asn Arg Ser Ser Thr Val Ser Phe Gly Asn His Phe Thr 1425 1430 1435 1440 He Leu Leu Lys Gly Leu Glu Lys Ser Ala Asp Leu Asn Gin Phe Pro 1445 1450 1455 Val Ser Leu Arg His Lys He Ser He Leu Asn Glu Asn Val He He 1460 1465 1470 Ala Leu Thr Asn Leu Ser Asn Ala Asn Val Asn Val Ser Leu Lys Phe 1475 1480 1485 Thr Leu Pro Met Gly Tyr Ser Pro Asn Lys Asp He Arg He Ala Phe 1490 1495 1500 Leu Arg Val Phe He Asp He Val Thr Asn Tyr Pro Val Asn Pro Glu 1505 1510 , 1515 1520 Lys His Glu Met Asp Lys Met Leu Ala He Asp Asp Phe Leu Lys Tyr 1525 1530 1535 He He Lys Asn Pro He Leu Ala Phe Phe Gly Ser Leu Ala Cys Ser 1540 1545 1550 Pro Ala Asp Val Asp Leu Tyr Ala Gly Gly Phe Leu Asn Ala Phe Asp 1555 1560 1565 Thr Arg Asn Ala Ser His He Leu Val Thr Glu Leu Leu Lys Gin Glu 1570 1575 1580 He Lys Arg Ala Ala Arg Ser Asp Asp He Leu Arg Arg Asn Ser Cys 1585 1590 1595 1600 Ala Thr Arg Ala Leu Ser Leu Tyr Thr Arg Ser Arg Gly Asn Lys Tyr 1605 1610 1615 Leu He Lys Thr Leu Arg Pro Val Leu Gin Gly He Val Asp Asn Lys 1620 1625 1630 Glu Ser Phe Glu He Asp Lys Met Lys Pro Gly Ser Glu Asn Ser Glu 1635 1640 1645 Lys Met Leu Asp Leu Phe Glu Lys Tyr Met Thr Arg Leu He Asp Ala 1650 1655 1660 He Thr Ser Ser He Asp Asp Phe Pro He Glu Leu Val Asp He Cys 1665 1670 1675 1680 Lys Thr He Tyr Asn Ala Ala Ser Val Asn Phe Pro Glu Tyr Ala Tyr 1685 1690 1695 He Ala Val Gly Ser Phe Val Phe Leu Arg Phe He Gly Pro Ala Leu 1700 1705 1710 Val Ser Pro Asp Ser Glu Asn He He He Val Thr His Ala His Asp 1715 1720 1725 Arg Lys Pro Phe He Thr Leu Ala Lys Val He Gin Ser Leu Ala Asn 1730 1735 1740 Gly Arg Glu Asn He Phe Lys Lys Asp He Leu Val Ser Lys Glu Glu 1745 1750 1755 1760 Phe Leu Lys Thr Cys Ser Asp Lys He Phe Asn Phe Leu Ser Glu Leu 1765 1770 1775 Cys Lys He Pro Thr Asn Asn Phe Thr Val Asn Val Arg Glu Asp Pro 1780 1785 1790 Thr Pro He Ser Phe Asp Tyr Ser Phe Leu His Lys Phe Phe Tyr Leu 1795 1800 1805 Asn Glu Phe Thr He Arg Lys Glu He He Asn Glu Ser Lys Leu Pro 1810 1815 1820 Gly Glu Phe Ser Phe Leu Lys Asn Thr Val Met Leu Asn Asp Lys He 1825 1830 1835 1840 Leu Gly Val Leu Gly Gin Pro Ser Met Glu He Lys Asn Glu He Pro 1845 1850 1855 Pro Phe Val Val Glu Asn Arg Glu Lys Tyr Pro Ser Leu Tyr Glu Phe 1860 1865 1870 Met Ser Arg Tyr Ala Phe Lys Lys Val Asp Met Lys Glu Glu Glu Glu 1875 1880 1885 Asp Asn Ala Pro Phe Val His Glu Ala Met Thr Leu Asp Gly He Gin 1890 1895 1900 He He Val Val Thr Phe Thr Asn Cys Glu Tyr Asn Asn Phe Val Met 1905 1910 1915 1920 Asp Ser Leu Val Tyr Lys Val Leu Gin He Tyr Ala Arg Met Trp Cys 1925 1930 1935 Ser Lys His Tyr Val Val He Asp Cys Thr Thr Phe Tyr Gly Gly Lys 1940 1945 1950 Ala Asn Phe Gin Lys Leu Thr Thr Leu Phe Phe Ser Leu He Pro Glu 1955 1960 1965 Gin Ala Ser Ser Asn Cys Met Gly Cys Tyr Tyr Phe Asn Val Asn Lys 1970 1975 1980 Ser Phe Met Asp Gin Trp Ala Ser Ser Tyr Thr Val Glu Asn Pro Tyr 1985 1990 1995 2000 Leu Val Thr Thr He Pro Arg Cys Phe He Asn Ser Asn Thr Asp Gin 2005 2010 2015 Ser Leu He Lys Ser Leu Gly Leu Ser Gly Arg Ser Leu Glu Val Leu 2020 2025 2030 Lys Asp Val Arg Val Thr Leu His Asp He Thr Leu Tyr Asp Lys Glu 2035 2040 2045 Lys Lys Lys Phe Cys Pro Val Ser Leu Lys He Gly Asn Lys Tyr Phe 2050 2055 2060 Gin Val Leu His Glu He Pro Gin Leu Tyr Lys Val Thr Val Ser Asn 2065 2070 2075 2080 Arg Thr Phe Ser He Lys Phe Asn Asn Val Tyr Lys He Ser Asn Leu 2085 2090 2095 He Ser Val Asp Val Ser Asn Thr Thr Gly Val Ser Ser Glu Phe Thr 2100 2105 2110 Leu Ser Leu Asp Asn Glu Glu Lys Leu Val Phe Cys Ser Pro Lys Tyr 2115 2120 2125 Leu Glu He Val Lys Met Phe Tyr Tyr Ala Gin Leu Lys Met Glu Glu 2130 2135 2140 Asp Phe Gly Thr Asp Phe Ser Asn Asp He Ser Phe Ser Thr Ser Ser 2145 2150 2155 2160 Ser Ala Val Asn Ala Ser Tyr Cys Asn Val Lys Glu Val Gly Glu He 2165 2170 2175 He Ser His Leu Ser Leu Val He Leu Val Gly Leu Phe Asn Glu Asp 2180 2185 2190 Asp Leu Val Lys Asn He Ser Tyr Asn Leu Leu Val Ala Thr Gin Glu 2195 2200 2205 Ala Phe Asn Leu Asp Phe Gly Thr Arg Leu His Lys Ser Pro Glu Thr 2210 2215 2220 Tyr Val Pro Asp Asp Thr Thr Thr Phe Leu Ala Leu He Phe Lys Ala 2225 2230 2235 2240 Phe Ser Glu Ser Ser Thr Glu Leu Thr Pro Tyr He Trp Lys Tyr Met 2245 2250 2255 Leu Asp Gly Leu Glu Asn Asp Val He Pro Gin Glu His He Pro Thr 2260 2265 2270 Val Val Cys Ser Leu Ser Tyr Trp Val Pro Asn Leu Tyr Glu His Val 2275 2280 2285 Tyr Leu Ala Asn Asp Glu Glu Gly Pro Glu Ala He Ser Arg He He 2290 2295 2300 Tyr Ser Leu He Arg Leu Thr Val Lys Glu Pro Asn Phe Thr Thr Ala 2305 2310 2315 2320 Tyr Leu Gin Gin He Trp Phe Leu Leu Ala Leu Asp Gly Arg Leu Thr 2325 2330 2335 Asn Val He Val Glu Glu He Val Ser His Ala Leu Asp Arg Asp Ser 2340 2345 2350 Glu Asn Arg Asp Trp Met Lys Ala Val Ser He Leu Thr Ser Phe Pro 2355 2360 2365 Thr Thr Glu He Ala Cys Gin Val He Glu Lys Leu He Asn Met He 2370 2375 2380 Lys Ser Phe Leu Pro Ser Leu Ala Val Glu Ala Ser Ala His Ser Trp 2385 2390 2395 2400 Ser Glu Leu Thr He Leu Ser Lys He Ser Val Ser He Phe Phe Glu 2405 2410 2415 Ser Pro Leu Leu Ser Gin Met Tyr Leu Pro Glu He Leu Phe Ala Val 2420 2425 2430 Ser Leu Leu He Asp Val Gly Pro Ser Glu He Arg Val Ser Leu Tyr 2435 2440 2445 Glu Leu Leu Met Asn Val Cys His Ser Leu Thr Asn Asn Glu Ser Leu 2450 2455 2460 Pro Glu Arg Asn Arg Lys Asn Leu Asp He Val Cys Ala Thr Phe Ala 2465 2470 2475 2480 Arg Gin Lys Leu Asn Phe He Ser Gly Phe Ser Gin Glu Lys Gly Arg 2485 2490 2495 Val Leu Pro Asn Phe Ala Ala Ser Ser Phe Ser Ser Lys Phe Gly Thr 2500 2505 2510 Leu Asp Leu Phe Thr Lys Asn He Met Leu Leu Met Glu Tyr Gly Ser 2515 2520 2525 He Ser Glu Gly Ala Gin Trp Glu Ala Lys Tyr Lys Lys Tyr Leu Met 2530 2535 2540 Asp Ala He Phe Gly His Arg Ser Phe Phe Ser Ala Arg Ala Met Met 2545 2550 2555 2560 He Leu Gly He Met Ser Lys Ser His Thr Ser Leu Phe Leu Cys Lys 2565 2570 2575 Glu Leu Leu Val Glu Thr Met Lys Val Phe Ala Glu Pro Val Val Asp 2580 2585 2590 Asp Glu Gin Met Phe He He He Ala His Val Phe Thr Tyr Ser Lys 2595 2600 2605 He Val Glu Gly Leu Asp Pro Ser Ser Glu Leu Met Lys Glu Leu Phe 2610 2615 2620 Trp Leu Ala Thr He Cys Val Glu Ser Pro His Pro Leu Leu Phe Glu 2625 2630 2635 2640 Gly Gly Leu Leu Phe Met Val Asn Cys Leu Lys Arg Leu Tyr Thr Val 2645 2650 2655 His Leu Gin Leu Gly Phe Asp Gly Lys Ser Leu Ala Lys Lys Leu Met 2660 2665 2670 Glu Ser Arg Asn Phe Ala Ala Thr Leu Leu Ala Lys Leu Glu Ser Tyr 2675 2680 2685 Asn Gly Cys He Trp Asn Glu Asp Asn Phe Pro His He He Leu Gly 2690 2695 2700 Phe He Ala Asn Gly Leu Ser He Pro Val Val Lys Gly Ala Ala Leu 2705 2710 2715 2720 Asp Cys Leu Gin Ala Leu Phe Lys Asn Thr Tyr Tyr Glu Arg Lys Ser 2725 2730 2735 Asn Pro Lys Ser Ser Asp Tyr Leu Cys Tyr Leu Phe Leu Leu His Leu 2740 2745 2750 Val Leu Ser Pro Glu Gin Leu Ser Thr Leu Leu Leu Glu Val Gly Phe 2755 2760 2765 Glu Asp Glu Leu Val Pro Leu Asn Asn Thr Leu Lys Val Pro Leu Thr 2770 2775 2780 Leu He Asn Trp Leu Ser Ser Asp Ser Asp Lys Ser Asn He Val Leu 2785 2790 2795 2800 Tyr Gin Gly Ala Leu Leu Phe Ser Cys Val Met Ser Asp Glu Pro Cys 2805 2810 2815 Lys Phe Arg Phe Ala Leu Leu Met Arg Tyr Leu Leu Lys Val Asn Pro 2820 2825 2830 He Cys Val Phe Arg Phe Tyr Thr Leu Thr Arg Lys Glu Phe Arg Arg 2835 2840 2845 Leu Ser Thr Leu Glu Gin Ser Ser Glu Ala Val Ala Val Ser Phe Glu 2850 2855 2860 Leu He Gly Met Leu Val Thr His Ser Glu Phe Asn Tyr Leu Glu Glu 2865 2870 2875 2880 Phe Asn Asp Glu Met Val Glu Leu Leu Lys Lys Arg Gly Leu Ser Val 2885 2890 2895 Val Lys Pro Leu Asp He Phe Asp Gin Glu His He Glu Lys Leu Lys 2900 2905 2910 Gly Glu Gly Glu His Gin Val Ala He Tyr Glu Arg Lys Arg Leu Ala 2915 2920 2925 Thr Met He Leu Ala Arg Met Ser Cys Ser 2930 2935 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3079 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ser Gin Pro Thr Lys Asn Lys Lys Lys Glu His Gly Thr Asp Ser 1 5 10 15 Lys Ser Ser Arg Met Thr Arg Thr Leu Val Asn His He Leu Phe Glu 20 25 30 . Arg He Leu Pro He Leu Pro Val Glu Ser Asn Leu Ser Thr Tyr Ser 35 40 45 Glu Val Glu Glu Tyr Ser Ser Phe He Ser Cys Arg Ser Val Leu He 50 55 60 Asn Val Thr Val Ser Arg Asp Ala Asn Ala Met Val Glu Gly Thr Leu 65 70 75 80 Glu Leu He Glu Ser Leu Leu Gin Gly His Glu He He Ser Asp Lys 85 90 95 Gly Ser Ser Asp Val He Glu Ser He Leu He He Leu Arg Leu Leu 100 105 110 Ser Asp Ala Leu Glu Tyr Asn Trp Gin Asn Gin Glu Ser Leu His Tyr 115 120 125 Asn Asp He Ser Thr His Val Glu His Asp Gin Glu Gin Lys Tyr Arg 130 135 140 Pro Lys Leu Asn Ser He Leu Pro Asp Tyr Ser Ser Thr His Ser Asn 145 150 155 160 Gly Asn Lys His Phe Phe His Gin Ser Lys Pro Gin Ala Leu He Pro 165 170 175 Glu Leu Ala Ser Lys Leu Leu Glu Ser Cys Ala Lys Leu Lys Phe Asn 180 185 190 Thr Arg Thr Leu Gin He Leu Gin Asn Met He Ser His Val His Gly 195 200 205 Asn He Leu Thr Thr Leu Ser Ser Ser He Leu Pro Arg His Lys Ser 210 215 220 Tyr Leu Thr Arg His Asn His Pro Ser His Cys Lys Met He Asp Ser 225 230 235 240 Thr Leu Gly His He Leu Arg Phe Val Ala Ala Ser Asn Pro Ser Glu 245 250 255 Tyr Phe Glu Phe He Arg Lys Ser Val Gin Val Pro Val Thr Gin Thr 260 265 270 His Thr His Ser His Ser His Ser His Ser Leu Pro Ser Ser Val Tyr 275 280 285 Asn Ser He Val Pro His Phe Asp Leu Phe Ser Phe He Tyr Leu Ser 290 295 300 Lys His Asn Phe Lys Lys Tyr Leu Glu Leu He Lys Asn Leu Ser Val 305 310 315 320 Thr Leu Arg Lys Thr He Tyr His Cys Leu Leu Leu His Tyr Ser Ala 325 330 335 Lys Ala He Met Phe Trp He Met Ala Arg Pro Ala Glu Tyr Tyr Glu 340 345 350 Leu Phe Asn Leu Leu Lys Asp Asn Asn Asn Glu His Ser Lys Ser Leu 355 360 365 Asn Thr Leu Asn His Thr Leu Phe Glu Glu He His Ser Thr Phe Asn 370 375 380 Val Asn Ser Met He Thr Thr Asn Gin Asn Ala His Gin Gly Ser Ser 385 390 395 400 Ser Pro Ser Ser Ser Ser Pro Ser Ser Pro Pro Ser Ser Ser Ser Ser 405 410 415 Asp Asn Asn Asn Gin Asn He He Ala Lys Ser Leu Ser Arg Gin Leu 420 425 430 Ser His His Gin Ser Tyr He Gin Gin Gin Ser Glu Arg Lys Leu His 435 440 445 Ser Ser Trp Thr Thr Asn Ser Gin Ser Ser Thr Ser Leu Ser Ser Ser 450 455 460 Thr Ser Asn Ser Thr Thr Thr Asp Phe Ser Thr His Thr Gin Pro Gly 465 470 475 480 Glu Tyr Asp Pro Ser Leu Pro Asp Thr Pro Thr Met Ser Asn He Thr 485 490 495 He Ser Ala Ser Ser Leu Leu Ser Gin Thr Pro Thr Pro Thr Thr Gin 500 505 510 Leu Gin Gin Arg Leu Asn Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 515 520 525 Ser Pro Ser Asn Ser Thr Pro Thr Gly Tyr Thr Ala Glu Gin Gin Ser 530 535 540 Arg Ala Ser Tyr Asp Ala His Lys Thr Gly His Thr Gly Lys Asp Tyr 545 550 555 560 Asp Glu His Phe Leu Ser Val Thr Arg Leu Asp Asn Val Leu Glu Leu 565 570 575 Tyr Thr His Phe Asp Asp Thr Glu Val Leu Pro His Thr Ser Val Leu 580 585 590 Lys Phe Leu Thr Thr Leu Thr Met Phe Asp He Asp Leu Phe Asn Glu 595 600 605 Leu Asn Ala Thr Ser Phe Lys Tyr He Pro Asp Cys Thr Met His Arg 610 615 620 Pro Lys Glu Arg Thr Ser Ser Phe Asn Asn Thr Ala His Glu Thr Gly 625 630 635 640 Ser Glu Lys Thr Ser Gly He Lys His He Thr Gin Gly Leu Lys Lys 645 650 655 Leu Thr Ser Leu Pro Ser Ser Thr Lys Lys Thr Val Lys Phe Val Lys 660 665 670 Met Leu Leu Arg Asn Leu Asn Gly Asn Gin Ala Val Ser Asp Val Ala 675 680 685 Leu Leu Asp Thr Met Arg Ala Leu Leu Ser Phe Phe Thr Met Thr Ser 690 695 700 Ala Val Phe Leu Val Asp Arg Asn Leu Pro Ser Val Leu Phe Ala Lys 705 710 715 720 Arg Leu He Pro He Met Gly Thr Asn Leu Ser Val Gly Gin Asp Trp 725 730 735 Asn Ser Lys He Asn Asn Ser Leu Met Val Cys Leu Lys Lys Asn Ser 740 745 750 Thr Thr Phe Val Gin Leu Gin Leu He Phe Phe Ser Ser Ala He Gin 755 760 765 Phe Asp His Glu Leu Leu Leu Ala Arg Leu Ser He Asp Thr Met Ala 770 775 780 Asn Asn Leu Asn Met Gin Lys Leu Cys Leu Tyr Thr Glu Gly Phe Arg 785 790 795 800 He Phe Phe Asp He Pro Ser Lys Lys Glu Leu Arg Lys Ala He Ala 805 810 815 Val Lys He Ser Lys Phe Phe Lys Thr Leu Phe Ser He He Ala Asp 820 825 830 He Leu Leu Gin Glu Phe Pro Tyr Phe Asp Glu Gin He Thr Asp He 835 840 845 Val Ala Ser He Leu Asp Gly Thr He He Asn Glu Tyr Gly Thr Lys 850 855 860 Lys His Phe Lys Gly Ser Ser Pro Ser Leu Cys Ser Thr Thr Arg Ser 865 870 875 880 Arg Ser Gly Ser Thr Ser Gin Ser Ser Met Thr Pro Val Ser Pro Leu 885 890 895 Gly Leu Asp Thr Asp He Cys Pro Met Asn Thr Leu Ser Leu Val Gly 900 905 910 Ser Ser Thr Ser Arg Asn Ser Asp Asn Val Asn Ser Leu Asn Ser Ser 915 920 925 Pro Lys Asn Leu Ser Ser Asp Pro Tyr Leu Ser His Leu Val Ala Pro 930 935 940 Arg Ala Arg His Ala Leu Gly Gly Pro Ser Ser He He Arg Asn Lys 945 950 955 960 He Pro Thr Thr Leu Thr Ser Pro Pro Gly Thr Glu Lys Ser Ser Pro 965 970 975 Val Gin Arg Pro Gin Thr Glu Ser He Ser Ala Thr Pro Met Ala He 980 985 990 Thr Asn Ser Thr Pro Leu Ser Ser Ala Ala Phe Gly He Arg Ser Pro 995 1000 1005 Leu Gin Lys He Arg Thr Arg Arg Tyr Ser Asp Glu Ser Leu Gly Lys 1010 1015 1020 Phe Met Lys Ser Thr Asn Asn Tyr He Gin Glu His Leu He Pro Lys 1025 1030 1035 1040 Asp Leu Asn Glu Ala Thr Leu Gin Asp Ala Arg Arg He Met He Asn 1045 1050 1055 He Phe Ser He Phe Lys Arg Pro Asn Ser Tyr Phe He He Pro His 1060 1065 1070 Asn He Asn Ser Asn Leu Gin Trp Val Ser Gin Asp Phe Arg Asn He 1075 1080 1085 Met Lys Pro He Phe Val Ala He Val Ser Pro Asp Val Asp Leu Gin 1090 1095 1100 Asn Thr Ala Gin Ser Phe Met Asp Thr Leu Leu Ser Asn Val He Thr 1105 1110 1115 1120 Tyr Gly Glu Ser Asp Glu Asn He Ser He Glu Gly Tyr His Leu Leu 1125 1130 1135 Cys Ser Tyr Thr Val Thr Leu Phe Ala Met Gly Leu Phe Asp Leu Lys 1140 1145 1150 He Asn Asn Glu Lys Arg Gin He Leu Leu Asp He Thr Val Lys Phe 1155 1160 1165 Met Lys Val Arg Ser His Leu Ala Gly He Ala Glu Ala Ser His His 1170 1175 1180 Met Glu Tyr He Ser Asp Ser Glu Lys Leu Thr Phe Pro Leu He Met 1185 1190 1195 1200 Gly Thr Val Gly Arg Ala Leu Phe Val Ser Leu Tyr Ser Ser Gin Gin 1205 1210 1215 Lys He Glu Lys Thr Leu Lys He Ala Tyr Thr Glu Tyr Leu Ser Ala 1220 1225 1230 He Asn Phe His Glu Arg Asn He Asp Asp Ala Asp Lys Thr Trp Val 1235 1240 1245 His Asn He Glu Phe Val Glu Ala Met Cys His Asp Asn Tyr Thr Thr 1250 1255 1260 Ser Gly Ser He Ala Phe Gin Arg Arg Thr Arg Asn Asn He Leu Arg 1265 1270 1275 1280 Phe Ala Thr He Pro Asn Ala He Leu Leu Asp Ser Met Arg Met He 1285 1290 1295 Tyr Lys Lys Trp His Thr Tyr Thr His Ser Lys Ser Leu Glu Lys Gin 1300 1305 1310 Glu Arg Asn Asp Phe Arg Asn Phe Ala Gly He Leu Ala Ser Leu Ser 1315 1320 1325 Gly He Leu Phe He Asn Lys Lys He Leu Gin Glu Met Tyr Pro Tyr 1330 1335 1340 Leu Leu Asp Thr Val Ser Glu Leu Lys Lys Asn He Asp Ser Phe He 1345 1350 1355 1360 Ser Lys Gin Cys Gin Trp Leu Asn Tyr Pro Asp Leu Leu Thr Arg Glu 1365 1370 1375 Asn Ser Arg Asp He Leu Ser Val Glu Leu His Pro Leu Ser Phe Asn 1380 1385 1390 Leu Leu Phe Asn Asn Leu Arg Leu Lys Leu Lys Glu Leu Ala Cys Ser 1395 1400 1405 Asp Leu Ser He Pro Glu Asn Glu Ser Ser Tyr Val Leu Leu Glu Gin 1410 1415 1420 He He Lys Met Leu Arg Thr He Leu Gly Arg Asp Asp Asp Asn Tyr 1425 1430 1435 1440 Val Met Met Leu Phe Ser Thr Glu He Val Asp Leu He Asp Leu Leu 1445 1450 1455 Thr Asp Glu He Lys Lys He Pro Ala Tyr Cys Pro Lys Tyr Leu Lys 1460 1465 1470 Ala He He Gin Met Thr Lys Met Phe Ser Ala Leu Gin His Ser Glu 1475 1480 1485 Val Asn Leu Gly Val Lys Asn His Phe His Val Lys Asn Lys Trp Leu 1490 1495 1500 Arg Gin He Thr Asp Trp Phe Gin Val Ser He Ala Arg Glu Tyr Asp 1505 1510 1515 1520 Phe Glu Asn Leu Ser Lys Pro Leu Lys Glu Met Asp Leu Val Lys Arg 1525 1530 1535 Asp Met Asp He Leu Tyr He Asp Thr Ala He Glu Ala Ser Thr Ala 1540 1545 1550 He Ala Tyr Leu Thr Arg His Thr Phe Leu Glu He Pro Pro Ala Ala 1555 1560 1565 Ser Asp Pro Glu Leu Ser Arg Ser Arg Ser Val He Phe Gly Phe Tyr 1570 1575 1580 Phe Asn He Leu Met Lys Gly Leu Glu Lys Ser Ser Asp Arg Asp Asn 1585 1590 1595 1600 Tyr Pro Val Phe Leu Arg His Lys Met Ser Val Leu Asn Asp Asn Val 1605 1610 1615 He Leu Ser Leu Thr Asn Leu Ser Asn Thr Asn Val Asp Ala Ser Leu 1620 1625 1630 Gin Phe Thr Leu Pro Met Gly Tyr Ser Gly Asn Arg Asn He Arg Asn 1635 1640 1645 Ala Phe Leu Glu Val Phe He Asn He Val Thr Asn Tyr Arg Thr Tyr 1650 1655 1660 Thr Ala Lys Thr Asp Leu Gly Lys Leu Glu Ala Ala Asp Lys Phe Leu 1665 1670 1675 1680 Arg Tyr Thr He Glu His Pro Gin Leu Ser Ser Phe Gly Ala Ala Val 1685 1690 1695 Cys Pro Ala Ser Asp He Asp Ala Tyr Ala Ala Gly Leu He Asn Ala 1700 1705 1710 Phe Glu Thr Arg Asn Ala Thr His He Val Val Ala Gin Leu He Lys 1715 1720 1725 Asn Glu He Glu Lys Ser Ser Arg Pro Thr Asp He Leu Arg Arg Asn 1730 1735 1740 Ser Cys Ala Thr Arg Ser Leu Ser Met Leu Ala Arg Ser Lys Gly Asn 1745 1750 1755 1760 Glu Tyr Leu He Arg Thr Leu Gin Pro Leu Leu Lys Lys He He Gin 1765 1770 1775 Asn Arg Asp Phe Phe Glu He Glu Lys Leu Lys Pro Glu Asp Ser Asp 1780 1785 1790 Ala Glu Arg Gin He Glu Leu Phe Val Lys Tyr Met Asn Glu Leu Leu 1795 1800 1805 Glu Ser He Ser Asn Ser Val Ser Tyr Phe Pro Pro Pro Leu Phe Tyr 1810 1815 1820 He Cys Gin Asn He Tyr Lys Val Ala Cys Glu Lys Phe Pro Asp His 1825 1830 1835 1840 Ala He He Ala Ala Gly Ser Phe Val Phe Leu Arg Phe Phe Cys Pro 1845 1850 1855 Ala Leu Val Ser Pro Asp Ser Glu Asn He He Asp He Ser His Leu 1860 1865 1870 Ser Glu Lys Arg Thr Phe He Ser Leu Ala Lys Val He Gin Asn He 1875 1880 1885 Ala Asn Gly Ser Glu Asn Phe Ser Arg Trp Pro Ala Leu Cys Ser Gin 1890 1895 1900 Lys Asp Phe Leu Lys Glu Cys Ser Asp Arg He Phe Arg Phe Leu Ala 1905 1910 1915 1920 Glu Leu Cys Arg Thr Asp Arg Thr He Asp He Gin Val Arg Thr Asp 1925 1930 1935 Pro Thr Pro He Ala Phe Asp Tyr Gin Phe Leu His Ser Phe Val Tyr 1940 1945 1950 Leu Tyr Gly Leu Glu Val Arg Arg Asn Val Leu Asn Glu Ala Lys His 1955 1960 1965 Asp Asp Gly Asp He Asp Gly Asp Asp Phe Tyr Lys Thr Thr Phe Leu 1970 1975 1980 Leu He Asp Asp Val Leu Gly Gin Leu Gly Gin Pro Lys Met Glu Phe 1985 1990 1995 2000 Ser Asn Glu He Pro He Tyr He Arg Glu His Met Asp Asp Tyr Pro 2005 2010 2015 Glu Leu Tyr Glu Phe Met Asn Arg His Ala Phe Arg Asn He Glu Thr 2020 2025 2030 Ser Thr Ala Tyr Ser Pro Ser Val His Glu Ser Thr Ser Ser Glu Gly 2035 2040 2045 He Pro He He Thr Leu Thr Met Ser Asn Phe Ser Asp Arg His Val 2050 2055 2060 Asp He Asp Thr Val Ala Tyr Lys Phe Leu Gin He Tyr Ala Arg He 2065 2070 2075 2080 Trp Thr Thr Lys His Cys Leu He He Asp Cys Thr Glu Phe Asp Glu 2085 2090 2095 Gly Gly Leu Asp Met Arg Lys Phe He Ser Leu Val Met Gly Leu Leu 2100 2105 2110 Pro Glu Val Ala Pro Lys Asn Cys He Gly Cys Tyr Tyr Phe Asn Val 2115 2120 2125 Asn Glu Thr Phe Met Asp Asn Tyr Gly Lys Cys Leu Asp Lys Asp Asn 2130 2135 2140 Val Tyr Val Ser Ser Lys He Pro His Tyr Phe He Asn Ser Asn Ser 2145 2150 2155 2160 Asp Glu Gly Leu Met Lys Ser Val Gly He Thr Gly Gin Gly Leu Lys 2165 2170 2175 Val Leu Gin Asp He Arg Val Ser Leu His Asp He Thr Leu Tyr Asp 2180 2185 2190 Glu Lys Arg Asn Arg Phe Thr Pro Val Ser Leu Lys He Gly Asp He 2195 2200 2205 Tyr Phe Gin Val Leu His Glu Thr Pro Arg Gin Tyr Lys He Arg Asp 2210 2215 2220 Met Gly Thr Leu Phe Asp Val Lys Phe Asn Asp Val Tyr Glu He Ser 2225 2230 2235 2240 Arg He Phe Glu Val His Val Ser Ser He Thr Gly Val Ala Ala Glu 2245 2250 2255 Phe Thr Val Thr Phe Gin Asp Glu Arg Arg Leu He Phe Ser Ser Pro 2260 2265 2270 Lys Tyr Leu Glu He Val Lys Met Phe Tyr Tyr Ala Gin He Arg Leu 2275 2280 2285 Glu Ser Glu Tyr Glu Met Asp Asn Asn Ser Ser Thr Ser Ser Pro Asn 2290 2295 2300 Ser Asn Asn Lys Val Lys Gin Gin Lys Glu Arg Thr He Leu Leu Cys 2305 2310 2315 2320 His Leu Leu Leu Val Ser Leu He Gly Leu Phe Asp Glu Ser Lys Lys 2325 2330 2335 Met Lys Asn Ser Ser Tyr Asn Leu He Ala Ala Thr Glu Ala Ser Phe 2340 2345 2350 Gly Leu Asn Phe Gly Ser His Phe His Arg Ser Pro Glu Val Tyr Val 2355 2360 2365 Pro Glu Asp Thr Thr Thr Phe Leu Gly Val He Gly Lys Ser Leu Ala 2370 2375 2380 Glu Ser Asn Pro Glu Leu Thr Ala Tyr Met Phe He Tyr Val Leu Glu 2385 2390 2395 2400 Ala Leu Lys Asn Asn Val He Pro His Val Tyr He Pro His Thr He 2405 2410 2415 Cys Gly Leu Ser Tyr Trp He Pro Asn Leu Tyr Gin His Val Tyr Leu 2420 2425 2430 Ala Asp Asp Glu Glu Gly Pro Glu Asn He Ser His He Phe Arg He 2435 2440 2445 Leu He Arg Leu Ser Val Arg Glu Thr Asp Phe Lys Ala Val Tyr Met 2450 2455 2460 Gin Tyr Val Trp Leu Leu Leu Leu Asp Asp Gly Arg Leu Thr Asp He 2465 2470 2475 2480 He Val Asp Glu Val He Asn His Ala Leu Glu Arg Asp Ser Glu Asn 2485 2490 2495 Arg Asp Trp Lys Lys Thr He Ser Leu Leu Thr Val Leu Pro Thr Thr 2500 2505 2510 Glu Val Ala Asn Asn He He Gin Lys He Leu Ala Lys He Arg Ser 2515 2520 2525 Phe Leu Pro Ser Leu Lys Leu Glu Ala Met Thr Gin Ser Trp Ser Glu 2530 2535 2540 Leu Thr He Leu Val Lys He Ser He His Val Phe Phe Glu Thr Ser 2545 2550 2555 2560 Leu Leu Val Gin Met Tyr Leu Pro Glu He Leu Phe He Val Ser Leu 2565 2570 2575 Leu He Asp Val Gly Pro Arg Glu Leu Arg Ser Ser Leu His Gin Leu 2580 2585 2590 Leu Met Asn Val Cys His Ser Leu Ala He Asn Ser Ala Leu Pro Gin 2595 2600 2605 Asp His Arg Asn Asn Leu Asp Glu He Ser Asp He Phe Ala His Gin 2610 2615 2620 Lys Val Lys Phe Met Phe Gly Phe Ser Glu Asp Lys Gly Arg He Leu 2625 2630 2635 2640 Gin He Phe Ser Ala Ser Ser Phe Ala Ser Lys Phe Asn He Leu Asp 2645 2650 2655 Phe Phe He Asn Asn He Leu Leu Leu Met Glu Tyr Ser Ser Thr Tyr 2660 2665 2670 Glu Ala Asn Val Trp Lys Thr Arg Tyr Lys Lys Tyr Val Leu Glu Ser 2675 2680 2685 Val Phe Thr Ser Asn Ser Phe Leu Ser Ala Arg Ser He Met He Val 2690 2695 2700 Gly He Met Gly Lys Ser Tyr He Thr Glu Gly Leu Cys Lys Ala Met 2705 2710 2715 2720 Leu He Glu Thr Met Lys Val He Ala Glu Pro Lys He Thr Asp Glu 2725 2730 2735 His Leu Phe Leu Ala He Ser His He Phe Thr Tyr Ser Lys He Val 2740 2745 2750 Glu Gly Leu Asp Pro Asn Leu Asp Leu Met Lys His Leu Phe Trp Phe 2755 2760 2765 Ser Thr Leu Phe Leu Glu Ser Arg His Pro He He Phe Glu Gly Ala 2770 2775 2780 Leu Leu Phe Val Ser Asn Cys He Arg Arg Leu Tyr Met Ala Gin Phe 2785 2790 2795 2800 Glu Asn Glu Ser Glu Thr Ser Leu He Ser Thr Leu Leu Lys Gly Arg 2805 2810 2815 Lys Phe Ala His Thr Phe Leu Ser Lys He Glu Asn Leu Ser Gly He 2820 2825 2830 Val Trp Asn Glu Asp Asn Phe Thr His He Leu He Phe He He Asn 2835 2840 2845 Lys Gly Leu Ser Asn Pro Phe He Lys Ser Thr Ala Phe Asp Phe Leu 2850 2855 2860 Lys Met Met Phe Arg Asn Ser Tyr Phe Glu His Gin He Asn Gin Lys 2865 2870 2875 2880 Ser Asp His Tyr Leu Cys Tyr Met Phe Leu Leu Tyr Phe Val Leu Asn 2885 2890 2895 Cys Asn Gin Phe Glu Glu Leu Leu Gly Asp Val Asp Phe Glu Gly Glu 2900 2905 2910 Met Val Asn He Glu Asn Lys Asn Thr He Pro Lys He Leu Leu Glu 2915 2920 2925 Trp Leu Ser Ser Asp Asn Glu Asn Ala Asn He Thr Leu Tyr Gin Gly 2930 2935 2940 Ala He Leu Phe Lys Cys Ser Val Thr Asp Glu Pro Ser Arg Phe Arg 2945 2950 2955 2960 Phe Ala Leu He He Arg His Leu Leu Thr Lys Lys Pro He Cys Ala 2965 2970 2975 Leu Arg Phe Tyr Ser Val He Arg Asn Glu He Arg Lys He Ser Ala 2980 2985 2990 Phe Glu Gin Asn Ser Asp Cys Val Pro Leu Ala Phe Asp He Leu Asn 2995 3000 3005 Leu Leu Val Thr His Ser Glu Ser Asn Ser Leu Glu Lys Leu His Glu 3010 3015 3020 Glu Ser He Glu Arg Leu Thr Lys Arg Gly Leu Ser He Val Thr Ser 3025 3030 3035 3040 Ser Gly He Phe Ala Lys Asn Ser Asp Met Met He Pro Leu Asp Val 3045 3050 3055 Lys Pro Glu Asp He Tyr Glu Arg Lys Arg He Met Thr Met He Leu 3060 3065 3070 Ser Arg Met Ser Cys Ser Ala 3075 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 870 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Lys Gly Trp Tyr His Gly Lys Leu Asp Arg Thr He Ala Glu Glu 1 5 10 15 Arg Leu Arg Gin Ala Gly Lys Ser Gly Ser Tyr Leu He Arg Glu Ser 20 25 30 Asp Arg Arg Pro Gly Ser Phe Val Leu Ser Phe Leu Ser Gin Met Asn 35 40 45 Val Val Asn His Phe Arg He He Ala Met Cys Gly Asp Tyr Tyr He 50 55 60 Gly Gly Arg Arg Phe Ser Ser Leu Ser Asp Leu He Gly Tyr Tyr Ser 65 70 75 80 His Val Ser Cys Leu Leu Lys Gly Glu Lys Leu Leu Tyr Pro Val Ala 85 90 95 Pro Pro Glu Pro Val Glu Asp Arg Arg Arg Val Arg Ala He Leu Pro 100 105 110 Tyr Thr Lys Val Pro Asp Thr Asp Glu He Ser Phe Leu Lys Gly Asp 115 120 125 Met Phe He Val His Asn Glu Leu Glu Asp Gly Trp Met Trp Val Thr 130 135 140 Asn Leu Arg Thr Asp Glu Gin Gly Leu He Val Glu Asp Leu Val Glu 145 150 155 160 Glu Val Gly Arg Glu Glu Asp Pro His Glu Gly Lys He Trp Phe His 165 170 175 Gly Lys He Ser Lys Gin Glu Ala Tyr Asn Leu Leu Met Thr Val Gly 180 185 190 Gin Val Cys Ser Phe Leu Val Arg Pro Ser Asp Asn Thr Pro Gly Asp 195 200 205 Tyr Ser Leu Tyr Phe Arg Thr Asn Glu Asn He Gin Arg Phe Lys He 210 215 220 Cys Pro Thr Pro Asn Asn Gin Phe Met Met Gly Gly Arg Tyr Tyr Asn 225 230 235 240 Ser He Gly Asp He He Asp His Tyr Arg Lys Glu Gin He Val Glu 245 250 255 Gly Tyr Tyr Leu Lys Glu Pro Val Pro Met Gin Asp Gin Glu Gin Val 260 265 270 Leu Asn Asp Thr Val Asp Gly Lys Glu He Tyr Asn Thr He Arg Arg 275 280 285 Lys Thr Lys Asp Ala Phe Tyr Lys Asn He Val Lys Lys Gly Tyr Leu 290 295 300 Leu Lys Lys Gly Lys Gly Lys Arg Trp Lys Asn Leu Tyr Phe He Leu 305 310 315 320 Glu Gly Ser Asp Ala Gin Leu He Tyr Phe Glu Ser Glu Lys Arg Ala 325 330 335 Thr Lys Pro Lys Gly Leu He Asp Leu Ser Val Cys Ser Val Tyr Val 340 345 350 Val His Asp Ser Leu Phe Gly Arg Pro Asn Cys Phe Gin He Val Val 355 360 365 Gin His Phe Ser Glu Glu His Tyr He Phe Tyr Phe Ala Gly Glu Thr 370 375 380 Pro Glu Gin Ala Glu Asp Trp Met Lys Gly Leu Gin Ala Phe Cys Asn 385 390 395 400 Leu Arg Lys Ser Ser Pro Gly Thr Ser Asn Lys Arg Leu Arg Gin Val 405 410 415 Ser Ser Leu Val Leu His He Glu Glu Ala His Lys Leu Pro Val Lys 420 425 430 His Phe Thr Asn Pro Tyr Cys Asn He Tyr Leu Asn Ser Val Gin Val 435 440 445 Ala Lys Thr His Ala Arg Glu Gly Gin Asn Pro Val Trp Ser Glu Glu 450 455 460 Phe Val Phe Asp Asp Leu Pro Pro Asp He Asn Arg Phe Glu He Thr 465 470 475 480 Leu Ser Asn Lys Thr Lys Lys Ser Lys Asp Pro Asp He Leu Phe Met 485 490 495 Arg Cys Gin Leu Ser Arg Leu Gin Lys Gly His Ala Thr Asp Glu Trp 500 505 510 Phe Leu Leu Ser Ser His He Pro Leu Lys Gly He Glu Pro Gly Ser 515 520 525 Leu Arg Val Arg Ala Arg Tyr Ser Met Glu Lys He Met Pro Glu Glu 530 535 540 Glu Tyr Ser Glu Phe Lys Glu Leu He Leu Gin Lys Glu Leu His Val 545 550 555 560 Val Tyr Ala Leu Ser His Val Cys Gly Gin Asp Arg Thr Leu Leu Ala 565 570 575 Ser He Leu Leu Arg He Phe Leu His Glu Lys Leu Glu Ser Leu Leu 580 585 590 Leu Cys Thr Leu Asn Asp Arg Glu He Ser Met Glu Asp Glu Ala Thr 595 600 605 Thr Leu Phe Arg Ala Thr Thr Leu Ala Ser Thr Leu Met Glu Gin Tyr 610 615 620 Met Lys Ala Thr Ala Thr Gin Phe Val His His Ala Leu Lys Asp Ser 625 630 635 640 He Leu Lys He Met Glu Ser Lys Gin Ser Cys Glu Leu Ser Pro Ser 645 650 655 Lys Leu Glu Lys Asn Glu Asp Val Asn Thr Asn Leu Thr His Leu Leu 660 665 670 Asn He Leu Ser Glu Leu Val Glu Lys He Phe Met Ala Ser Glu He 675 680 685 Leu Pro Pro Thr Leu Arg Tyr He Tyr Gly Cys Leu Gin Lys Ser Val 690 695 700 Gin His Lys Trp Pro Thr Asn Thr Thr Met Arg Thr Arg Val Val Ser 705 710 715 720 Gly Phe Val Phe Leu Arg Leu He Cys Pro Ala He Leu Asn Pro Arg 725 730 735 Met Phe Asn He He Ser Asp Ser Pro Ser Pro He Ala Ala Arg Thr 740 745 750 Leu He Leu Val Ala Lys Ser Val Gin Asn Leu Ala Asn Leu Val Glu 755 760 765 Phe Gly Ala Lys Glu Pro Tyr Met Glu Gly Val Asn Pro Phe He Lys 770 775 780 Ser Asn Lys His Arg Met He Met Phe Leu Asp Glu Leu Gly Asn Val 785 790 795 800 Pro Glu Leu Pro Asp Thr Thr Glu His Ser Arg Thr Asp Leu Ser Arg 805 810 815 Asp Leu Ala Ala Leu His Glu He Cys Val Ala His Ser Asp Glu Leu 820 825 830 Arg Thr Leu Ser Asn Glu Arg Gly Ala Gin Gin His Val Leu Lys Lys 835 840 845 Leu Leu Ala He Thr Glu Leu Leu Gin Gin Lys Gin Asn Gin Tyr Thr 850 855 860 Lys Thr Asn Asp Val Arg 865 870 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 766 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Schizosaccharomyces pombe (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : Met Thr Lys Arg His Ser Gly Thr Leu Ser Ser Ser Val Leu Pro Gin 1 5 10 15 Thr Asn Arg Leu Ser Leu Leu Arg Asn Arg Glu Ser Thr Ser Val Leu 20 25 30 Tyr Thr He Asp Leu Asp Met Glu Ser Asp Val Glu Asp Ala Phe Phe 35 40 45 His Leu Asp Arg Glu Leu His Asp Leu Lys Gin Gin He Ser Ser Gin 50 55 60 Ser Lys Gin Asn Phe Val Leu Glu Arg Asp Val Arg Tyr Leu Asp Ser 65 70 75 80 Lys He Ala Leu Leu He Gin Asn Arg Met Ala Gin Glu Glu Gin His 85 90 95 Glu Phe Ala Lys Arg Leu Asn Asp Asn Tyr Asn Ala Val Lys Gly Ser 100 105 110 Phe Pro Asp Asp Arg Lys Leu Gin Leu Tyr Gly Ala Leu Phe Phe Leu 115 120 125 Leu Gin Ser Glu Pro Ala Tyr He Ala Ser Leu Val Arg Arg Val Lys 130 135 140 Leu Phe Asn Met Asp Ala Leu Leu Gin He Val Met Phe Asn He Tyr 145 150 155 160 Gly Asn Gin Tyr Glu Ser Arg Glu Glu His Leu Leu Leu Ser Leu Phe 165 170 175 Gin Met Val Leu Thr Thr Glu Phe Glu Ala Thr Ser Asp Val Leu Ser 180 185 190 Leu Leu Arg Ala Asn Thr Pro Val Ser Arg Met Leu Thr Thr Tyr Thr 195 200 205 Arg Arg Gly Pro Gly Gin Ala Tyr Leu Arg Ser He Leu Tyr Gin Cys 210 215 220 He Asn Asp Val Ala He His Pro Asp Leu Gin Leu Asp He His Pro 225 230 235 240 Leu Ser Val Tyr Arg Tyr Leu Val Asn Thr Gly Gin Leu Ser Pro Ser 245 250 255 Glu Asp Asp Asn Leu Leu Thr Asn Glu Glu Val Ser Glu Phe Pro Ala 260 265 270 Val Lys Asn Ala He Gin Glu Arg Ser Ala Gin Leu Leu Leu Leu Thr 275 280 285 Lys Arg Phe Leu Asp Ala Val Leu Asn Ser He Asp Glu He Pro Tyr 290 295 300 Gly He Arg Trp Val Cys Lys Leu He Arg Asn Leu Thr Asn Arg Leu 305 310 315 320 Phe Pro Ser He Ser Asp Ser Thr He Cys Ser Leu He Gly Gly Phe 325 330 335 Phe Phe Leu Arg Phe Val Asn Pro Ala He He Ser Pro Gin Thr Ser 340 345 350 Met Leu Leu Asp Ser Cys Pro Ser Asp Asn Val Arg Lys Thr Leu Ala 355 360 365 Thr He Ala Lys He He Gin Ser Val Ala Asn Gly Thr Ser Ser Thr 370 375 380 Lys Thr His Leu Asp Val Ser Phe Gin Pro Met Leu Lys Glu Tyr Glu 385 390 395 400 Glu Lys Val His Asn Leu Leu Arg Lys Leu Gly Asn Val Gly Asp Phe 405 410 415 Phe Glu Ala Leu Glu Leu Asp Gin Tyr He Ala Leu Ser Lys Lys Ser 420 425 430 Leu Ala Leu Glu Met Thr Val Asn Glu He Tyr Leu Thr His Glu He 435 440 445 He Leu Glu Asn Leu Asp Asn Leu Tyr Asp Pro Asp Ser His Val His 450 455 460 Leu He Leu Gin Glu Leu Gly Glu Pro Cys Lys Ser Val Pro Gin Glu 465 470 475 480 Asp Asn Cys Leu Val Thr Leu Pro Leu Tyr Asn Arg Trp Asp Ser Ser 485 490 495 He Pro Asp Leu Lys Gin Asn Leu Lys Val Thr Arg Glu Asp He Leu 500 505 510 Tyr Val Asp Ala Lys Thr Leu Phe He Gin Leu Leu Arg Leu Leu Pro 515 520 525 Ser Gly His Pro Ala Thr Arg Val Pro Leu Asp Leu Pro Leu He Ala 530 535 540 Asp Ser Val Ser Ser Leu Lys Ser Met Ser Leu Met Lys Lys Gly He 545 550 555 560 Arg Ala He Glu Leu Leu Asp Glu Leu Ser Thr Leu Arg Leu Val Asp 565 570 575 Lys Glu Asn Arg Tyr Glu Pro Leu Thr Ser Glu Val Glu Lys Glu Phe 580 585 590 He Asp Leu Asp Ala Leu Tyr Glu Arg He Arg Ala Glu Arg Asp Ala 595 600 605 Leu Gin Asp Val His Arg Ala He Cys Asp His Asn Glu Tyr Leu Gin 610 615 620 Thr Gin Leu Gin He Tyr Gly Ser Tyr Leu Asn Asn Ala Arg Ser Gin 625 630 635 640 He Lys Pro Ser His Ser Asp Ser Lys Gly Phe Ser Arg Gly Val Gly 645 650 655 Val Val Gly He Lys Pro Lys Asn He Lys Ser Ser Asn Thr Val Lys 660 665 670 Leu Ser Ser Gin Gin Leu Lys Lys Glu Ser Val Leu Leu Asn Cys Thr 675 680 685 He Pro Glu Phe Asn Val Ser Asn Thr Tyr Phe Thr Phe Ser Ser Pro 690 695 700 Ser Thr Asp Asn Phe Val He Ala Val Tyr Gin Arg Gly His Ser Lys 705 710 715 720 Val Leu Val Glu Val Cys He Cys Leu Asp Asp Val Leu Gin Arg Arg 725 730 735 Tyr Ala Ser Asn Pro Val Val Asp Leu Gly Phe Leu Thr Phe Glu Ala 740 745 750 Asn Lys Leu Tyr His Leu Phe Glu Gin Leu Phe Leu Arg Lys 755 760 765

Claims: WHAT IS CLAIMED IS: 1. A method of blocking a Ras-induced effect on a cell, comprising a step of introducing a GTPase Activating (GAP) protein to said cell. 2. A method of Claim 1, wherein said Ras is an oncogenic Ras. 3. A method of Claim 1, wherein said Raε substantially lacks GTPase activity. 4. A method of Claim 1, wherein said effect is induction of cell proliferation or transformation. 5. A method of treating an oncogenic Raε transformed cell comprising the step of introducing to said cell a GAP protein capable of εuppreεεing the transformation of said cell. 6. The method of either Claim 1 or 5, wherein said cell is a eukaryotic cell, including a mammalian cell, including a human cell. 7. The method of either Claim 1 or 5, wherein said step of introducing is by expresεion of a nucleic acid encoding said GAP protein. 8. A method for the manufacture of a pharmaceutical composition for treating an oncogenic Ras transformed cell comprising admixing a GAP protein capable of suppreεεing the transformation of said cell with a pharmaceutically acceptable carrier. 9. The method of any of Claims l,5or 8 wherein said GAP protein binds to said Ras protein with a Kd of lesε than 200 nM. 10. The method of any of Claims 1,5 or 8 wherein said GAP protein is selected from the group of: a) a fragment of a mammalian GAP protein; b) a fragment of a mammalian NFl-GRD protein; c) a homologue or mimetic of a or b; and d) the proteinε defined by SEQ ID NO: 1 or SEQ ID NO: 2. 11. The method of any of Claimε 1, 5 or 8 wherein εaid GAP protein iε selected from the group of: a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and b) a fragment of a mutant mammalian GAP protein having a εequence with an amino acid substitution at a position corresponding to a poεition 1063 through 1651 of NFl or the correεponding region of other GAP proteinε. 12. A method of Claim 11, wherein said subεtitution is a conservative substitution. 13. The method of any of Claims 1, 5 or 8 wherein said GAP protein interacts with Ras and blockε interaction of an effector molecule which bindε to Ras at a position from 32 to 40 or from 59 to 65. 14. A method of Claim 2, wherein said GAP protein does not block signal transduction of non-oncogenic Ras. 15. A method of either of Claim 1 or 2, further comprising the steps of identifying the responsible oncogenic Ras and selecting said GAP protein which blockε εaid identified oncogenic Raε. 16. A method of identifying appropriate GAP proteins useful for treating a mutated Ras-induced condition of a eukaryote cell comprising the stepε of : a) identifying the mutated Raε which induceε εaid condition; and b) screening various GAP variants for specific variants which are capable of blocking said condition. 17. A method of Claim 16, wherein said eukaryote cell is a mammalian cell, including a human cell. 18. A method of 16, further comprising additional screening to determine which GAP variants have minimal effect on non-mutated Ras effects. 19. A GAP protein capable of blocking transformation of a cell, where said transformation is due to an oncogenic Ras. 20. A protein of Claim 19, wherein said GAP is selected from the group of: a) a fragment of a mammalian GAP protein; b) a fragment of a mammalian NFl-GRD protein; c) a homologue or mimetic of a or b; and d) a protein defined by SEQ ID NO: 1 or SEQ ID NO: 2. 21. A protein of Claim 19, selected from the group of: a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid subεtitution at a position corresponding to a position from 1063 through 1651 or the corresponding region of other GAP proteins. 22. A protein of Claim 21, wherein said substitution is a conservative substitution. 23. A protein of Claim 19, wherein said protein interacts with Ras and blocks interaction of an effector molecule which binds to Ras at a position from 32 to 40 or from 59 to 65. 24. A protein of Claim 19, wherein said cell is a eukaryotic cell, including a mammalian cell, including a human cell. 25. A protein of Claim 19, wherein said oncogenic Ras substantially lackε GTPaεe activity. 26. A protein of Claim 19, which binds to said Ras protein with a Kd of less than 200 nM. 27. A protein of Claim 19, wherein said protein interferes with interaction of Ras*GTP with an effector compound. 28. An iεolated nucleic acid encoding a protein normally expreεεed aε a protein of Claim 19. 29. A pharmaceutical compoεition for treating an oncogenic Raε tranεformed cell compriεing a GAP protein capable of suppressing the transformation of said cell and a pharmaceutically carrier. 30. The pharmaceutical compoεition of claim 29 wherein the GAP protein binds to said Raε protein with a Kd of leεε than 200 nM. 31. The pharmaceutical compoεition of claim 29 wherein said GAP protein is selected from the group of: a) a fragment of a mammalian GAP protein; b) a fragment of a mammalian NFl-GRD protein; c) a homologue or mimetic of a or b; and d) the proteins defined by SEQ ID NO: 1 or SEQ ID NO: 2. 32. The pharmaceutical composition of claim 29 wherein said GAP protein is selected from the group of: a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position 1063 through 1651 of NFl or the corresponding region of other GAP proteins. 33. The pharmaceutical composition of claim 29 wherein said GAP protein interacts with Ras and blocks interaction of an effector molecule which binds to Ras at a position from 32 to 40 or from 59 to 65. 34. The use of a GAP protein capable of suppressing the transformation of an oncogenic Ras transformed cell and a pharmaceutically carrier for treating said oncogenic Ras transformed cell. 35. The use of a GAP protein capable of suppressing the transformation of an oncogenic Ras transformed cell for the manufacture of a medicament for treating said oncogenic Ras transformed cell. 36. The use of either Claim 34 or 35 in which the GAP protein binds to said Ras protein with a Kd of lesε than 200 nM. 37. The uεe of either Claim 34 or 35 in which the GAP protein iε εelected from the group of: a) a fragment of a mammalian GAP protein; b) a fragment of a mammalian NFl-GRD protein; c) a homologue or mimetic of a or b; and d) the proteins defined by SEQ ID NO: 1 or SEQ ID NO: 2 38. The use of either Claim 34 or 35 in which the GAP protein is selected from the group of: a) a fragment of a mammalian GAP protein having a wild type sequence, including a human GAP protein; and b) a fragment of a mutant mammalian GAP protein having a sequence with an amino acid substitution at a position corresponding to a position 1063 through 1651 of NFl or the corresponding region of other GAP proteins. 39. The use of either Claim 34 or 35 in which the GAP protein interacts with Ras and blocks interaction of an effector molecule which binds to Ras at a position from 32 to 40 or from 59 to 65.

Inventors: KAZIRO YOSHITO, NAKAFUKU MASATO, KAZIRO, YOSHITO, NAKAFUKU, MASATO
Applicants: KAZIRO YOSHITO, NAKAFUKU MASATO, SCHERING CORP, KAZIRO, YOSHITO, NAKAFUKU, MASATO, SCHERING CORPORATION
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, A61P35, A61P43, C07K14, C12N15, C12P21, C12R1, G01N33